Novel surface protein of the malaria parasite plasmodium falciparum

ABSTRACT

Vaccines, antibodies, polypeptides, DNAs and RNAs for diagnosis, prophylaxis, treatment and detection of malaria or Plasmodium infection.  P. falciparum  antigen and fragments thereof and recombinant proteins or fusion proteins produced thereby. Methods for diagnosis, prophylaxis, treatment and detection of malaria or Plasmodium infection.

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. ProvisionalApplication Ser. No. 60/293,633, filed May 25, 2001, the content ofwhich is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] This invention concerns vaccines, antibodies, proteins, DNAs andRNAs for diagnosis, prophylaxis and treatment of Plasmodium infectionsand for detection of Plasmodium. In particular, this invention concernsP. falciparum antigen comprised of a protein, as well as polyclonal andmonoclonal antibodies directed against the antigen, DNA and RNA encodingthe P. falciparum antigen and fragments and analogs thereof, and methodsfor production of recombinant or fusion proteins. This invention alsoconcerns methods for diagnosis, prophylaxis, treatment of P. falciparuminfections and detection of P. falciparum.

BACKGROUND OF THE INVENTION

[0003]Plasmodium falciparum is the most virulent etiological agent ofhuman malaria, responsible for over 90% of mortality due to the disease.Each year 300-500 million people are infected by malaria parasites,resulting in 1.5-3 million deaths (World Health Organization (1998) FactSheet N94, World Health Organization, Geneva, Switzerland). Efforts toeradicate malaria generally have failed, and currently the disease isendemic in more than 90 countries throughout the tropics. Widespread andincreasing drug and insecticide resistance have exacerbated thesituation, undermining the effectiveness of existing malaria controlmethods that depend on chemotherapy and vector control, respectively.Novel means to fight the disease are needed urgently, and a vaccine ispredicted to have the greatest impact in addition to being the mostcost-effective control measure (Miller, L. H., and Hoffman, S. L. (1998)Nat. Med. 4, 520-524).

[0004] Experimental support for the development of a vaccine for humanmalaria was provided first by the use of radiation-attenuatedsporozoites as immunogens (Clyde, D. F., et al. (1973) Am. J. Med. Sci.266, 169-177). The success of this experimental vaccination provided theimpetus for the search for mechanisms of protective immune responses andthe target antigens involved.

[0005] The circumsporozoite (CS) protein was identified as the majorsurface antigen of Plasmodium sporozoites (Yoshida, N., et al. (1980)Science 207, 71-73; Zavala, F., et al. (1983) J. Exp. Med. 157,1947-1957; Dame, J. B., et al. (1984) Science 225, 593-599). The CSprotein has been a leading vaccine candidate antigen because irradiatedsporozoite-induced, protected human volunteers have high titers ofanti-CS antibodies (Herrington, D., et al. (1991) Am. J. Trop. Med. Hyg.5, 539-547), and CS-specific monoclonal antibodies and cytotoxicT-lymphocytes could adoptively transfer protection in a rodent malariamodel system (Weiss, W. R., et al. (1992) J. Immunol. 149, 2103-2109).However, attempts to induce protection in humans using P. falciparumCS-based vaccines, despite recent improvement in their immunogenicity,have repeatedly yielded only partial success (Ballou, W. R., et al.(1987) Lancet 1, 1277-1281; Herrington, D., et al. (1987) Nature 328,257-259; Hoffman, S. L., et al. (1993) in Molecular ImmunologicConsiderations in Malaria Vaccine Development, (Good, M. F., and Saul,A. J., eds) pp. 149-167, CRC Press, London; Stoute, J. A., et al. (1997)N. Engl. J. Med. 336, 86-91; Stoute, J. A., et al. (1998) J. Infect.Dis. 178, 1139-1144). The inability to develop a vaccine based on the CSprotein has been interpreted to indicate that additional antigens play arole in irradiated sporozoite-mediated protection against infection(Galey, B., et al. (1990) Infect. Immun. 9, 2995-3001).

[0006] The SPf66 vaccine was the first multicomponent P. falciparumvaccine developed that contains peptide epitopes derived from a numberof erythrocytic stage antigens (Patarroyo, M. E., et al. (1988) Nature332, 158). These peptide epitopes were synthesized chemically and linkedtogether by the repeat amino acid sequence of the pre-erythrocytic stageCS antigen. Although the SPf66 vaccine showed promising results in earlytrials, large-scale human trials revealed later that it provides onlylimited protection (Alonso, P. L., et al. (1994) Lancet 344, 1175;Alonso, P. L., et al. (1994) Vaccine 12, 181; D'Alessandro, U., et al.(1995) The Lancet 346, 462).

[0007] More recently, another multistage, multicomponent P. falciparumvaccine, NYVAC-Pf7, was developed (Tine, J. A., et al. (1996) Infectionand Immunity 64, 3833). This vaccine formulation combined seven antigensthat are known to be expressed in different developmental stages of theP. falciparum lifecycle. NYVAC-Pf7 was tested in human volunteers anddisappointingly did not protect against malaria. Of the 35 volunteerschallenged, only one did not develop malaria, although there was asignificant delay in the onset of parasitemia in the remainingvolunteers (Ockenhouse, C. F., et al. (1998) Journal of InfectiousDisease 177, 1664). Interestingly, as with previous malaria vaccines,among all vaccinated individuals, protection or delay in the prepatentperiod did not correlate with antibody titers, CTL activity, orlymphoproliferative responses.

[0008] The failure of single-antigen CS-based and the multicomponentvaccines SPf66 and NYVAC-Pf7 may indicate that the vaccines lackessential epitopes required to induce the protective immunity that is asconsistent and long-lasting as that observed in theirradiated-sporozoite vaccine.

[0009] Thus, there is a continuous need to identify antigens that mayact independently, additively or synergistically with the CS protein inthe development of a multicomponent vaccine.

[0010] Additionally, there is a need to have available methods forreproducible expression of specific target for P. falciparum antigen inlarge amounts, which antigen would provide a better immunogen. Thisapproach requires that a specific P. falciparum antigen is cloned andidentified as a potential candidate through its ability to elicit anantibody response that is immunoprotective. Before antibodies producedin this manner are tested in or administered to humans or animals, invitro testing of their inhibitory effect on P. falciparum in culturedcells and in vivo studies would be desirable.

SUMMARY OF THE INVENTION

[0011] One aspect of this invention concerns vaccines, antigens,antibodies, proteins, DNAs and RNAs for prophylaxis, treatment anddetection or diagnosis of malaria or Plasmodium infections.

[0012] Another aspect of this invention concerns a P. falciparum antigenprotein MB2 and fragments thereof.

[0013] Still another aspect of this invention concerns polyclonal ormonoclonal antibodies directed against the P. falciparum antigen.

[0014] Still yet another aspect of this invention concerns DNA and RNAencoding the P. falciparum antigen and fragments thereof.

[0015] Still another aspect of this invention concerns a natural,synthetic or recombinant vaccine useful for active immunization ofanimals and humans against P. falciparum infection.

[0016] Still another aspect of this invention concerns a natural,synthetic or recombinant protein useful for preparation of passiveimmune products for treatment of established infection.

[0017] Another aspect of this invention concerns a natural, synthetic orrecombinant DNA vaccine capable of endogenous production of inhibitoryamount of anti-P. falciparum antibodies.

[0018] Another aspect of this invention concerns a natural, synthetic orrecombinant RNA vaccine capable of endogenous development of inhibitoryamount of anti-P. falciparum antibodies.

[0019] Still yet another aspect of the invention is the use of antigen,antibody, DNA or RNA to detect the presence of MB2 or antibodies to MB2,or DNA or RNA encoding MB2, for diagnosis in a human or animal host ordetection in the environment.

[0020] Another aspect of this invention concerns the sequence of a 1610amino acid protein of MW 120 kDa present in sporozoites and merozoites,and its amino acid and size variants.

[0021] Another aspect of this invention concerns the DNA sequence of4830 nucleotides encoding the 120 kDa protein, its nucleotide and sizevariants and its upstream regulatory elements.

[0022] Another aspect of this invention concerns the RNA sequencedetermined by the DNA sequence of MB2 and its nucleotide and sizevariants including polyadenylation sequence.

[0023] Still yet another aspect of this invention concerns a group ofMB2 recombinant or expressed protein targets of polyclonal antibodieswhich inhibit P. falciparum infection, invasion, or adhesion.

[0024] Another aspect of this invention concerns a method forprophylaxis and treatment of malaria or Plasmodium infections usingvaccines, antibodies, proteins, DNAs and RNAs of the invention.

[0025] Still yet another aspect of this invention concerns a method ofprophylaxis, treatment, inhibition or retardation of malaria or aPlasmodium infection comprising administering to a subject in need ofsuch treatment an amount of an anti-P. falciparum polyclonal ormonoclonal antibodies prophylactically or therapeutically effective toprovide immunity against infection or treatment for disease.

[0026] Still yet another aspect of this invention concerns a method ofprophylaxis, treatment, retardation, or inhibition of malaria orPlasmodium infection comprising administering to a subject in need ofsuch treatment a vaccine comprising the polypeptide of this invention orits DNA or RNA capable of endogenous stimulation of the production ofinhibitory amount of anti-P. falciparum antibodies or protectivecellular immune responses.

[0027] Still yet another aspect of this invention concerns a method fordiagnosing Plasmodium infection of a subject, comprising steps: (a)contacting a body specimen, fluid or tissue obtained from the subjectwith an anti-P. falciparum monoclonal or polyclonal antibody; and (b)detecting the formation of antibody-antigen complex wherein the presenceof the complex indicates the presence of a P. falciparum organism in thesubject.

[0028] Still yet another aspect of this invention concerns a method fordetecting anti-P. falciparum antibody in a subject, said methodcomprising steps: (a) contacting a body specimen, fluid or tissueobtained from the subject with the MB2; and (b) detecting a formation ofantibody-antigen complex wherein the presence of the complex indicatesthe presence of a P. falciparum antibody in the subject.

[0029] Still another aspect of this invention is a P. falciparumdiagnostic or detection kit comprising anti-P. falciparum specificmonoclonal and polyclonal antibodies or antigen according to theinvention and a means for detection of an antibody-antigen complex.

[0030] Yet another aspect of the invention pertains to reagentsresulting from activation of a cell mediated immune response to MB2antigen, including cytokines and cytotoxic cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]FIG. 1. Southern and Northern analyses of the MB2 gene andtranscription product. A, Southern blot of P. falciparum genomic DNA,strain FCR3, digested with various restriction enzymes and hybridizedwith the spz-MB2 cDNA clone. Lane 1, EcoRI/HindIII; lane 2,PstI/HindIII; lane 3, PstI/EcoRI; lane 4, PstI/EcoRV; lane 5, PstI/NdeI.B, Northern blot of P. falciparum blood-stage mRNA hybridized with aprobe derived from nucleotides 1-580 of the MB2 ORF. The lane contained20 μg of total RNA. The approximate locations of molecular size markersin kilobases (kb) are indicated to the right of each of the panels.

[0032]FIG. 2. Structure of the MB2 gene and expression products. A,schematic representation of cDNA and genomic clones used to identify andassemble a cDNA containing the complete ORF of the MB2 gene. The cDNAs,spz-MB2, c3-1-18, c18-4-23, and c3-4-29, are represented as horizontallines above a linear representation of the full-length MB2 cDNA. Thenumbers above each cDNA refer to the terminal nucleotide positions inthe completed cDNA. The As in parentheses in the cDNA clones representthe internal and terminal priming poly(A) sites of the oligo(dT)primers. The full-length cDNA is represented as a horizontal linenumbered with the positions of the translation initiation (ATG) andtranslation termination (TAA) codons, and the beginning and end of thesequence. The 5′ end untranslated region (5′-UTR) and polyadenylationsequences (A) also are indicated. The three horizontal lines at thebottom denote the MB2 genomic clones, g2-6-8, g2-4-4#5, and g6-2-2. Thelocations of the terminal nucleotides with respect to the cDNA areindicated above each line. Four horizontal arrows (a-d) represent theorientation and approximate location of gene amplification primers usedto verify the contiguity of the sequence in the parasite genome. B,schematic representation of the MB2 protein sequence. The three domains,basic (B), acidic (A), and GTP-binding (G), are indicated as blocks withthe junctions of the domains numbered below. The amino (H₂N) andcarboxyl (COOH) ends are labeled. The four short horizontal linesrepresent the approximate extents of the polypeptides, MB2-B, MB2-C,MB2-FA, and MB2-IF2, used to generate antibodies. C, primary amino acidsequence of the conceptual translation of the MB2 gene. Amino acids inbold represent the putative signal peptide; bold and boxed, putativenuclear localization sequences; bold and italicized, repeat regions witha single repeat unit underlined; bold and underlined, cell-surfaceretention sequence; italicized and boxed, motifs conserved in the Gdomain.

[0033]FIG. 3. Immunolocalization of the MB2 protein in differentdevelopmental stages of P. falciparum. A-D: sporozoite preparations. Ashows a cross-section of a sporozoite (S) in the mosquito salivary gland(Sg) reacted with anti-B domain antiserum. B is a cross-section of afree sporozoite reacted with anti-A domain antiserum. C and D arepartial-oblique and cross sections (respectively) of sporozoites inHepG2-A16 cells (He) reacted with anti-B and anti-A domain antisera,respectively. PV is the parasitophorous vacuole space. E-G: asexualstage parasite preparations. E is a cross-section of a trophozoite in anerythrocyte (E), showing localization principally to the parasitenucleus (N) and some in the parasite cytoplasm (Pc). F is a section ofschizonts showing MB2 localization to the nucleus and some cytoplasm.Hemazoin (Hz) also is visible. Both E and F were reacted with anti-Bdomain antiserum. G shows sections of parasites at the merozoite (Mz)stage reacted with anti-A domain antiserum, showing only cytoplasmiclocalization. H and I: localization of MB2 in gametocytes (labeled G)reacted with anti-B and anti-A domain antisera, respectively. MB2 can bedetected in the nucleus, cytoplasm, and the PV space. Arrows indicatethe location of gold particles. J: localization of MB2 in theexoerythrocytic (EE) stages of an Aotus monkey hepatocyte (AH) reactedwith anti-B domain antiserum. All bars are 0.5 μm in length.

[0034]FIG. 4. Immunoblot analysis of protein extracts of P. falciparumsporozoite and blood stages. A and B: protein extracts prepared fromsporozoites recovered from salivary glands of infected mosquitoes. C andD: proteins extracts prepared from asexual blood-stage parasites. A andC were probed with anti-B domain (MB2-B) antiserum; B and D were probedwith anti-A domain (MB2-FA) antiserum. The molecular size markers (inkDa) are indicated to the left of each figure, and arrows to the rightmark the locations of the MB2 polypeptides.

[0035]FIG. 5. Summary of expression and localization data for the MB2protein. Each panel (A-D) lists the stage of the parasite (first line)and the molecular size determination based on immunoblotting (secondline). The third line indicates which domains were detected in eitherthe immunoblotting or immune electron microscopy experiments. The immuneelectron micrographs are excerpted from FIG. 4. The asterisks in B and Dindicate that the molecular size is not confirmed by immunoblottinganalyses. The question mark (?)in D indicates that the presence of the Gdomain could not be unequivocally confirmed. All abbreviations are as inFIGS. 3 and 4.

[0036]FIG. 6. Immunoblot analyses to assess the antigenicity of MB2recombinant peptides. A: Schematic representation of the MB2 proteinsequence. The three domains, Basic (B), Acidic (A), and GTP-binding (G),are indicated as blocks with the amino acid junctions numbered below.The seven short horizontal lines represent the approximate extent ofeach of the polypeptides that were expressed as GST-fusion recombinantproteins. B: Immunoblots of GST-MB2 recombinant proteins reacted withanti-GST rabbit serum (Anti-GST); serum of a protected volunteer (#5volunteer); or serum of a person living in a malaria-endemic area(Endemic serum). Immunoblots were prepared in triplicate, and each lanecontains 50-100 ng of purified GST-MB2 recombinant proteins. Recombinantproteins MB2-C, MB2-D and MB2-FA, listed in bold letters, contain aminoacid repeats. Approximate molecular weights of the fusion proteins areindicated in kilodaltons (kDa).

[0037]FIG. 7. Amino acid sequence alignment showing the sizepolymorphism in the repeat region of the MB2 gene from differentlaboratory strains and field isolates. Amino acid positions 211 to 264make up the repeat domain. Identical amino acids outside the repeatdomain are not shown. Field isolates were surveyed from India, Venezuela(Ven), Thailand (Thai) and Papua New Guinea (PNG).

[0038] Abbreviations

[0039] The abbreviations used herein include: CS, circumsporozoite; CRS,cell-surface retention signal; IEM, immunoelectron microscopy; GST,glutathione S-transferase; NLS, nuclear localization signal; ORF, openreading frame; PV, parasitophosporous vacuole; aa, amino acid(s); UTR,untranslated region; bp, base pair(s); MSP, merozoite surface protein;TRAP, thrombospondin related anonymous protein.

DETAILED DESCRIPTION OF THE INVENTION

[0040] A novel P. falciparum gene, MB2, was identified by screening asporozoite cDNA library with the serum of a human volunteer protectedexperimentally by the bites of P. falciparum-infected and irradiatedmosquitoes. The single-exon, single-copy MB2 gene is predicted to encodea protein with an M_(r) of 187,000. The MB2 protein has anamino-terminal basic domain, a central acidic domain, and acarboxyl-terminal domain with similarity to the GTP-binding domain ofthe prokaryotic translation initiation factor 2. MB2 is expressed insporozoites, the liver, and blood-stage parasites and gametocytes. TheMB2 protein is distributed as an ˜120-kDa moiety on the surface ofsporozoites and is imported into the nucleus of blood-stage parasites asan ˜66-kDa species. Proteolytic processing is favored as the mechanismregulating the distinct subcellular localization of the MB2 protein.This differential localization provides multiple opportunities toexploit the MB2 gene product as a vaccine or therapeutic target.

[0041] MB2 elicited an immune response detected by serum antibodies inall human volunteers (5/5) that were immunized experimentally andprotected by the bites of infected and irradiated mosquitoes. Incontrast, no anti-MB2 antibodies were detected in the serum of allirradiated-sporozoite immunized but not protected volunteers (3/3).Anti-MB2 antibodies also were detected in the sera of 83% of theindividuals living in a malaria endemic area of Kenya.

[0042] Protected volunteers produced antibodies that recognizedpreferentially a region (b) of the basic domain (B) of MB2 that does notcontain repeat sequences of amino acids. In contrast, naturally-infectedindividuals produced antibodies that recognize preferentially regions ofMB2 that contain amino acid repeats. Furthermore, anti-MB2 antibodiesagainst the non-repeat region of the B domain inhibited to a greaterextent the invasion in vitro of a hepatoma cell line by sporozoites thandid antibodies against regions that contain amino acid repeats.

[0043] Sequence analysis of 11 field isolates and four laboratorystrains showed that the antigenic region of the B domain of the MB2 geneis conserved absolutely in parasites obtained from different parts ofthe world.

[0044] The molecular and immunogenic properties of MB2 indicate that itis a likely vaccine candidate and drug target for malaria. Recombinantantigens derived from genes whose products are localized to the surfaceare potential vaccine candidate molecules for eliciting protectiveimmunity. In addition, proteins that localize to the nucleus arepotential drug targets. The MB2 gene product has both of theseproperties.

[0045] MB 2 was discovered using a process whereby subtractivehybridization was used in conjunction with specific cDNA libraries touncover cDNAs and genes that correspond to novel surface antigens onsporozoites, including sporozoites of P. falciparum. The screeningprocedures combine a process of subtractive hybridization to removecDNAs corresponding to the immunodominant circumsporozoite protein froma sporozoite library, as well as the screening of the library with theserum of a person protected by the bite of irradiated, P.falciparum-infected Anopheles gambiae.

[0046] This molecular approach allows the identification of novelsurface proteins on sporozoites. Prior to this approach, the discoveryof non-CS genes expressed in sporozoites was fortuitous. The presentinventors have overcome the abundance of CS genes by using thesubtractive procedures, allowing the identification of non-CS clones.

I. The MB2 P. falciparum Antigen

[0047] The MB2 gene is a single-exon, single-copy gene predicted toencode a protein with an M_(r) of 187,000. MB2 is expressed insporozoites, the liver, and blood-stage parasites and gametocytes. TheMB2 protein is found on the surface of sporozoites and is imported intothe nucleus of blood-stage parasites.

[0048] Strategy for discovering novel surface proteins on sporozoites ofthe human malaria parasite P. falciparum. The present inventorsconstructed cDNA libraries from PolyA⁺ RNA isolated from sporozoites(spz) of P. falciparum. The approach was to generate from this spzlibrary a sublibrary following subtractive hybridization procedures toremove all cDNAs with sequence homology to the CS gene, according to themethod described in Example 1.

[0049] To evaluate the efficiency of CS depletion in the subtracted cDNAlibrary, 100 ng of the recovered single-stranded cDNA were amplifiedwith oligonucleotide primers complementary to the cloning vector togenerate a heterogeneous mixture of fragments of about 200 to about 1000bp in size. The products were amplified again with gene-specificprimers. Although two marker genes, TRAP (Robson, K. J., et al. (1988)Nature 335, 79-82; Rogers, W. O., et al. (1992) Proc. Natl. Acad. Sci.U.S. A. 89, 9176-9180) and P2 (Fidock, D. A., et al. (1998) Exp.Parasitol. 89, 125-128) were detected, no signal corresponding to the CSgene was amplified, indicating that the subtraction technique was highlyefficient (data not shown). The amplification products were subclonedinto the UniZap vector and packaged in λ phage, producing a library of1.45×10⁶ primary phage.

[0050] Discovery of MB2, a gene encoding a novel protein present insporozoites of P. falciparum. The CS-depleted cDNA library was screenedwith the polyclonal serum isolated from a volunteer who had beenprotected against P. falciparum by the bites of irradiated, infectedmosquitoes, according to the method of Example 2. These proceduresallowed the identification of cDNAs corresponding to novel surfaceproteins. A screening of 1×10⁴ primary phage with the human volunteerserum led to the selection of 18 candidate phage clones. These werere-screened, and 12 phage again were reactive for antibodies.

[0051] Southern analyses showed that 1 of the 12 secondary cloneshybridized specifically to P. falciparum genomic DNA and showed patternsof hybridization consistent with a single-copy gene (FIG. 1A). This496-bp sporozoite cDNA clone was designated spz-MB2 and was selected forfurther characterization.

[0052] Northern analyses of RNA isolated from blood-stage parasitescultured in vitro and hybridized with probes derived from both the 5′and 3′ ends of the complete ORF (described below) produced a singlepositive signal at ˜7.5 kilobases (FIG. 1B).

[0053] Sequence analysis of the MB2 cDNA and gene. A comparison of thesize of the spz-MB2 cDNA with the mRNA detected in the Northern analysesindicates that it is not a full-length cDNA. Furthermore, primarysequencing of spz-MB2 showed that it lacked a translation terminationcodon and represented an incomplete ORF.

[0054] Sequence complementary to MB2 was detected in an asexualblood-stage cDNA library using specific gene amplification primers, andtherefore the library was screened with the spz-MB2 cDNA. Twooverlapping blood-stage cDNAs, c3-1-18 and c18-4-23, were identified(FIG. 2A).

[0055] Nucleotide sequence analysis revealed that the reading frame ofspz-MB2 was contained entirely within a contig formed by these twocDNAs. The c3-1-18 clone contained a putative translation initiationcodon and a 435-bp 5′ end untranslated region (UTR). The 3′ end terminiof the c3-1-18 and c18-4-23 cDNAs each have what appear to bepolyadenylation (poly(A)) sequences characteristic of the 3′ end terminiof processed mRNAs. However, there were no translation terminationcodons located to the 5′ end of the poly (A) tracks in either of thecDNAs, and the overlap of c3-1-18 with c18-4-23 revealed that the 17terminal A nucleotides in c3-1-18 comprise an internal A-rich nucleotidestretch in c18-4-23. Therefore, it was concluded that the oligo(dT)primed the mRNA for cDNA synthesis from within the coding region.

[0056] To obtain additional 3′ end sequence of MB2, a Sau3AI genomiclibrary (strain ITO) was screened using as a probe the 400 nucleotidesat the 3′ end of c18-4-23. A genomic clone, g2-4-4#5, was identifiedhaving overlapping and contiguous sequence with c18-4-23. The sequenceof g2-4-4#5 confirmed that the 17-A region at the 3′ end of c18-4-23 isan internal A-rich nucleotide track, supporting the conclusion thatthese A-rich internal nucleotide tracks were primed by oligo(dT).

[0057] To obtain additional 3′ end cDNA sequence, the 600 nucleotides atthe 3′ end of g2-4-4#5 were used to screen the blood-stage cDNA library,resulting in the identification of the cDNA clone, c3-4-29. Sequencingof c3-4-29 revealed that it was contiguous with c18-4-23. In addition,there are three stop codons at the 3′ end of c-3-4-29, commencing atnucleotides 5266, 5272, and 5293, and there is a putative poly(A) regionnear the 3′ end of the last stop codon.

[0058] The positions of the stop codons and the authenticity of thepoly(A) of the MB2 cDNA were supported by the genomic clone, g6-2-2,identified by screening the genomic library with a probe derived fromthe 3′ end of c3-4-29. Similarly, the 5′ end UTR and the start codon ofMB2 also were verified by the clone g2-6-8, isolated from the genomiclibrary using a probe derived from the 5′ end of c3-1-18.

[0059] The overlapping primary sequences of the three blood-stage cDNAclones and the contiguity of their reading frames allowed the assemblyof a complete ORF of MB2 that is 4,830 nucleotides in length, of which77%. of the bases are A-T pairs. No nucleotide polymorphisms wereobserved among the cDNA and genomic sequences, indicating that there isa single allele of the MB2 gene encoded and expressed in the parasitestrains used in our analyses.

[0060] Because the nucleotide sequences of the three genomic clones didnot overlap, we designed gene amplification primers a, b, c, and d(shown in FIG. 2A and described in Example 3) to assess the contiguityof the MB2 gene in the parasite genome. Amplification products producedby the primer pairs a+b and c+d with parasite genomic DNA as thetemplate gave the predicted product sizes, ˜700 and ˜1000 bp,respectively, indicating that MB2 is organized as a contiguous,single-exon gene in the parasite genome.

[0061] Sequence analysis of the MB2 putative translation product. MB2encodes a putative translation product that is 1610 amino acids (aa) inlength with an approximate molecular mass of 187 kDa (FIGS. 2, B and C).The predicted protein is rich in asparagine (15%) and lysine (13%) andis strongly basic with a calculated net charge of +20 at pH 7 and a pIof 8.3.

[0062] The primary amino acid sequence can be separated into threedistinct, linear domains, the first of which is an amino-terminal basicdomain of 490 residues (aa 1-490) with a calculated net charge of +30and a pI of 9.4. This has been designated the “B” domain. This domaincontains a region of six 9-aa imperfect repeats (aa 211-264) with theconsensus sequence L, N, S, K, K, N, D/N, N, T/S.

[0063] The central acidic domain, designated “A,” encompasses 496residues (aa 491-986) with a calculated net charge of 26.2 and a pI of6.1. The boundary between the B and A domains was selected to maximizethe basic and acidic properties of the respective domains. The A domaincontains two regions of imperfect repeats of 5 amino acids. The firstregion (aa 493-542) contains 10 repeats with a consensus sequence of D,N, Q/P, N, Y. The second region (aa 870-914) contains nine repeats witha consensus of I/M, N/D, V, Q, D. No similarities to any sequences ofknown function deposited in the data bases were detected for either theB or A domains.

[0064] Finally, a 624-residue carboxyl-terminal domain (aa 987-1610)with sequence similarity to the GTP-binding domain of the prokaryotictranslation initiation factor 2 (IF2), as revealed by the BLAST searchprogram (Altschul, S. F., et al. (1997) Nucleic Acids Res. 25,3389-3402), has been designated “G”. The boundary between the A and Gdomains was selected based on the start of the regions of similarity ofthe MB2 protein with known IF2 molecules.

[0065] In contrast to its overall hydrophilic nature, the MB2polypeptide contains at the amino terminus a strongly hydrophobic region(aa 1-25) mapped by a Kyte-Doolittle hydrophobicity plot. The PSORTcomputer program (Nakai, K., and Kanehisa, M. (1992) Genomics 14,897-911) predicted an uncleavable signal peptide in the hydrophobicamino-terminal region of MB2. However, the SignalP program (Nielsen, H.,et al. (1997) Protein Eng. 10, 1-6) predicted that the signal peptidecould be cleaved between a pair of S—S residues at aa 27-28 (FIG. 2C).Currently, there is no experimental data to support one alternative overthe other.

[0066] The PSORT program also predicted a number of nuclear localizationsignals (NLS), PKKK (aa 120-123), RRKK (aa 173-176), KKKKK (aa 652-656),and a bipartite NLS, KKNKELPFNN-KFKKIIK (aa 718-734), within the B and Adomains. Multiple putative sites for N-glycosylation, N-myristoylation,and phosphorylation were detected by the ScanProsite program (Appel, R.D., Bairoch, A., and Hochstrasser, D. F. (1994) Trends Biochem. Sci. 19,258-260; data not shown).

[0067] There is a polybasic motif, KKKKKGKSRKK (aa 956-966), just beforethe start of the G domain, that could function as a plasma membranelocalization signal as well as a cell-surface retention sequence (CRS)(Hancock, J. F., Paterson, H., and Marshall, C. J. (1990) Cell 63,133-139). This sequence also could be a putative NLS, although PSORTfailed to identify it as such. The similarity of the G domain to theGTP-binding domains of the prokaryotic IF2 proteins includes theconservation of sequence and spacing of three motifs, GX₄GK (aa999-1005), DX₂K (aa 1046-1049), and NKXD (aa 1100-1104), common to thisfamily of proteins (Dever, T. E., Glynias, M. V., and Merrick, W. C.(1987) Proc. Natl. Acad. Sci. U.S. A. 84, 1814-1818). There is a smallvariation in the third motif, TKXD, in MB2 as compared with theconsensus seen in other G proteins (FIG. 2C).

[0068] Ultrastructural localization of MB2 protein. Immunoelectronmicroscopy (IEM) was used to study the subcellular localization of theMB2 antigen. All rabbit antisera prepared against recombinant peptidesderived from the B and A domains (FIG. 2A), and reacted with sectionedmaterial containing sporozoites, showed that MB2 protein was localizedpredominantly to the surface (FIGS. 3, A-D). This was true ofsporozoites in salivary glands (FIGS. 3, A and B), as well as those thatinvaded in vitro cells of the human liver cell line, HepG2-A16 (FIGS. 3,C and D). No antibody reaction was detected with sporozoites using theanti-G domain antibody (data not shown). Preimmune control sera for allreagents were negative (data not shown).

[0069] In contrast, the majority of the MB2 protein detected inblood-stage parasites using both antisera against the B domain waslocalized in the nucleus, with some antibody reactivity detected in thecytoplasm (FIGS. 3, E and F) and data not shown). Rabbit antiseraagainst the A and G domains detected protein only in the cytoplasm ofthese parasites (FIG. 3G and data not shown). Furthermore, the numbersof gold particles observed in sections of parasites exposed toantibodies against the A and G domains were low when compared with thesignal produced by the anti-B domain antisera, suggesting that themajority of MB2 protein present at the blood stages does not contain theA and G domains.

[0070] MB2 protein was detected in the cytoplasm, nucleus, andparasitophorous vacuole (PV) space of gametocyte-stage parasites usingthe anti-B domain antiserum (FIG. 3H). MB2 protein detected by theanti-A domain antiserum was localized only in the PV space (FIG. 3I),indicating that the protein detected in the nucleus and cytoplasm withthe anti-B domain antiserum does not contain the A domain. The anti-Gdomain antiserum produced a high background signal, making it difficultto interpret any specific localization pattern.

[0071] Finally, IEM was used to investigate the localization of MB2protein in the exoerythrocytic stages of the parasite. A section of theliver of an infected Aotus monkey was reacted with the anti-B domainantiserum. Although it is difficult to locate the parasites in thesesections, some of the protein was shown to be localized mostly in thecytoplasm with some in the PV space (FIG. 3J). Sections reacted with theanti-A domain antiserum had high backgrounds obscuring any evidence of aspecific localization pattern.

[0072] Immunoblot analyses to determine the relative size of the MB2protein. A series of immunoblotting experiments were performed withparasite protein extracts prepared from the sporozoite and blood stagesto determine the relative size of the MB2 protein. The results are shownin FIG. 4. Both anti-B and anti-A domain antisera detected a singlepolypeptide of ˜120 kDa at the sporozoite stage (FIGS. 4, A and B). Noimmunoblot analyses were done on sporozoite preparations with anti-Gdomain antiserum because of the negative results obtained in the IEManalyses.

[0073] Immunoblotting using anti-B domain antibody detected a singlepolypeptide of ˜66 kDa at the blood stages (FIG. 4C). Furthermore, the66-kDa polypeptide was not detected with either the anti-A or anti-Gdomain antibodies (FIG. 4D and data not shown). These data areconsistent with the IEM study and indicate that the MB2 polypeptidelocated at the surface of sporozoites consists of only the B and Adomains, and the polypeptide translocated into the nucleus of parasitesat the blood stages consists primarily of the B domain.

[0074] Discussion of MB2 antigen. The Southern analyses shows that MB2is present in the parasite genome most likely as a single-copy gene. Thenucleotide sequence data indicates that it consists of a single exon andis represented most likely in the examined parasite samples by a singleallele. The Northern analyses with mRNA obtained from the blood-stageparasites indicates that MB2 is expressed as a single large transcript.It is not known if this size is common to the other developmental stagesof the parasite because of the difficulty in obtaining sufficient mRNAfor blotting experiments. However, because the gene is single-copy andcontains no intron, it is unlikely that multiple transcripts areproduced, resulting either from expression of different genes oralternative splicing of a single gene. Thus, it is likely that thesingle-transcript expression of MB2 is common to other developmentalstages.

[0075] The overall length of the reconstructed MB2 cDNA is 2.2 kilobasessmaller than the RNA species detected in the Northern analyses. Thisdifference likely results from large 5′ end, and perhaps 3′ end,untranslated regions. Although no single genomic nor cDNA clone wasidentified that spans the entire ORF of MB2, the overlapping primarysequence of the cDNA clones, the contiguity of their reading frames, andthe gene amplification analyses of the genomic clones indicate that thecomplete expressed sequence of the MB2 gene has been identified.

[0076] The complete ORF of MB2 predicts a full-length protein of 187kDa. However, there are many predicted sites for post-translationalmodification by myristoylation, glycosylation and phosphorylation.Therefore, it is likely that the actual molecular weight of the primaryprotein structure is increased by processing of individual amino acids.The predicted MB2 protein is rich in asparagine (15%) and lysine (13%),and therefore is strongly basic. Asparagine is the most commonly used(˜12%) amino acid in P. falciparum, followed by lysine and glutamic acid(˜10%) (Hyde, J. E., and Sims, P. F. (1987) Gene 61, 177-187; Weber, J.L. (1987) Gene (Amst.) 52, 103-109). Two other sporozoite surfaceproteins, CS and the sporozoite-threonine-and-asparagine-rich protein(STARP) (Fidock, D. A., et al. (1994) Mol. Biochem. Parasitol. 64,219-232), contain 29% and 25% asparagine, respectively. It has beensuggested that asparagine-rich motifs in the amino acid sequence mightbe targets of opsonizing antibodies, promoting parasite phagocytosis byimmune cells (Gysin, J., et al. (1993) J. Immunol. Methods 159, 209-219;Barale, J. C., et al. (1997) Mol. Biochem. Parasitol. 87, 169-181;Barale, J. C., et al. (1997) Infect. Immun. 65, 3003-3010). Whether theMB2 protein is a target of opsonizing antibodies is not known, but it isrecognized by the immune serum of a human volunteer protected by theirradiated sporozoite vaccine.

[0077] Unlike a number of characterized Plasmodium genes that are activeonly in certain stages, the MB2 gene is expressed in many developmentalstages of the parasite life cycle. However, the MB2 gene product hasdifferential localization throughout development. The stage-dependentdifferential localization of the MB2 protein suggests strongly that ithas a multifunctional role during development of the parasites. It isconceivable that it functions as a signal recognition molecule while itis on the surface of the sporozoites. It then may transmit a signal tothe nucleus by migrating there during the blood stages. Once inside thenucleus, it may function in the regulation of gene expression,participating in the process of turning off genes that are not requiredand activating genes that are required for blood stage infection.Examples of genes that are known to be inactivated as the parasitedevelops to the blood-stage are CS (Suhrbier, A., et al. (1988) Eur. J.Cell Biol. 46, 25-30; Atkinson, C. T., et al. (1989) Am. J. Trop. Med.Hyg. 41, 9-17) and TRAP, and genes that are activated are merozoitesurface protein genes, MSPs (Smythe, J. A., et al. (1988) Immunology 85,5195-5199).

[0078] In gametocytes, the protein product is localized in the nucleus,cytoplasm, and the PV space. This differential localization may indicatethat the MB2 gene product is in a transitional phase from its functionalrole in the nucleus to the cell surface or it may have a role in thedevelopment of the sexual stages of the parasite. In the exoerythrocyticstage, the MB2 protein detectable by anti-B domain antisera is localizedmainly in the cytoplasm, although some can be detected in the PV space.As with the gametocytes, this expression may be a transitional phase inthe specific localization as the parasite develops in the liver. Theexpression of MB2 in this stage is important potentially as a vaccinetarget since the hepatocyte expresses major histocompatibility complexmolecules that can be recognized by T cells. Research in the last 10years has indicated that the infected hepatocyte can be an importanttarget for immune attack.

[0079] Although the MB2 protein is localized on the surface membrane ofsporozoites, the primary amino acid sequence contains no apparenttransmembrane domain or glycosylphosphatidylinositol anchor signal.However, the amino acid sequence does contain a polybasic motif that wasshown to function as plasma membrane localization signal as well as CRSmotif (Hancock, supra; Lokeshwar, V. B., Huang, S. S., and Huang, J. S.(1990) J. Biol. Chem. 265, 1665-1675). Many cytokines are retained onthe membrane surface of the producer cell in a process mediated by theCRS. Studies have shown that, if the basic amino acids are deleted ormutated to acidic or neutral amino acids, then the membrane localizationof the protein is affected (Ostman, A., et al. (1991) Cell Regul. 2,503-512; Cadwallader, K. A., et al. (1994) Mol. Cell. Biol. 14,4722-4730). Therefore, the polybasic motif is the most likely domain tobe used to localize the MB2 product to the membrane surface ofsporozoites.

[0080] Data suggests that protein processing is the mechanism by whichMB2 regulates its differential cellular localization (FIG. 5). The MB2protein is localized mostly to the surface membrane of sporozoites as an˜120-kDa species consisting of the B and A domains (FIGS. 3 (A and B)and 5A) It is noted that the predicted molecular mass of the B and Adomains combined is ˜116 kDa. This similarity in molecular mass betweenthe actual protein and the predicted domains is remarkably close anddoes not take into consideration the effects of post-translationalchanges to specific amino acids or the arbitrary boundaries assigned tothe domains.

[0081] As the sporozoites invade hepatocytes represented by the humancultured liver cells, the B and A domains of the MB2 protein are stilldetectable on the membrane surface (FIGS. 3C and D) and 5B). There is noimmunoblot data for this stage, but it can be inferred from the IEMstudy that the size of the protein is most likely ˜120 kDa, similar tothe size detected in free sporozoites.

[0082] As the parasite develops to the blood stages, the majority of MB2protein detected inside the parasite nucleus consists only of the Bdomain and is represented in immunoblots by an ˜66-kDa species (FIGS. 3(E and F) and 5C). The predicted molecular mass, ˜57 kDa, of the Bdomain selected by analysis of the amino acid primary structure isconsistent with this smaller size polypeptide.

[0083] As the parasite differentiates to gametocytes, the MB2 protein isfound in the PV space as well as the nucleus and cytoplasm (FIGS. 3 (Hand I) and 5D). Based on the different labeling patterns seen with theanti-B and anti-A domain antisera, it is likely that the signal in thecytoplasm and nucleus originates from the ˜66-kDa moiety. The protein inthe PV space contains at least the B and A domains, and may contain theG domain. However, as noted in the results, the IEM study using theanti-G domain antibody is inconclusive, and there is no immunoblot datathat would provide the size of MB2 for the gametocyte stage.

[0084] The MB2 protein detected weakly in the cytoplasm of blood-stageparasites by the anti-A and anti-G domain antisera most likelyrepresents the full-length, newly synthesized MB2 protein that has notbeen processed proteolytically into the ˜66-kDa polypeptide. The datasuggests that the full-length MB2 protein is processed specifically atthe sporozoite stage to the ˜120-kDa polypeptide during synthesis and/orcellular trafficking. The ˜120-kDa species contains the polybasicCRS-like motif, allowing it to be preferentially retained on the surfaceof the sporozoite. The secondary or higher-order structure of the˜120-kDa protein may conceal the NLS in the B and A domains.

[0085] At the blood stage, the full-length MB2 protein is processedspecifically into the ˜66-kDa polypeptide as supported by the absence ofthe ˜120-kDa species. The processing of the MB2 protein into the ˜66-kDapolypeptide would remove the polybasic motif, thus removing the membranetargeting signal, and perhaps this processing exposes the nuclearlocalization signals allowing the ˜66-kDa polypeptide to translocate tothe nucleus. Finally, MB2 protein in the gametocyte stage may beprocessed into at least two forms, one of which consists of at least theB and A domains and is exported to the PV space. The other form,consisting most likely of only the B domain, is transported to thenucleus.

[0086] Another interesting feature of the MB2 protein is the G domain,which has significant sequence similarity to the prokaryotic IF2. Thedata presented herein indicates that the G domain is not present in theMB2 protein detected on the sporozoite surface, nor is it present in thenucleus at the blood stages. It is conceivable that the cleavage of theMB2 protein requires energy, and this requirement is fulfilled by the Gdomain since it can bind to GTP. The cleavage process most likelyincludes removal of the G domain as evidenced by the inability to detectit with specific antiserum in most stages of the parasite.Alternatively, because MB2 can bind potentially to GTP, it is possiblethat there are conformational differences between the GTP-bound,GDP-bound, and unbound states that can regulate the distinct proteolyticprocessing of the MB2 protein.

II. Immunological Characterization P. falciparum Sporozoite SurfaceAntigen MB2.

[0087] The MB2 antigen is a target of antibody response in protected butnot unprotected volunteers exposed to the bites of P. falciparuminfected and irradiated mosquitoes. The MB2 antigen possesses intriguingimmunogenic and molecular properties that indicate that it may be animportant immune target for vaccine studies.

[0088] Recombinant protein expression and purification. GST-MB2recombinant proteins representing various regions of the coding sequenceof MB2 were expressed in bacteria, in accordance with the methoddescribed in Example 5. The seven MB2 recombinant proteins, MB2-A,MB2-B, MB2-C, MB2-D, MB2-E, MB2-FA and MB2-IF2 are shown schematicallyin FIG. 6A. (Recombinant proteins MB2-B, MB2-C, MB2-FA and MB2-IF2 arealso shown schematically in FIG. 2B.)

[0089] Bacteria transformed with plasmids carrying different regions ofMB2 required different induction times and culture media for optimalyield, as shown in Table 1. The yield also varied greatly, with someregions being expressed well in bacteria, while others expressed poorly.The inclusion of about 80 to about 85 mM of imidazole in the washingbuffer and the overnight wash during isolation, as described in Example5, was found to be important in improving the purity of the recombinantprotein. All of the expressed proteins were soluble and thus purifiedeasily from the cell-free bacterial lysates by one-step nickel columnchromatography. TABLE 1 Optimal conditions to express GST-MB2recombinant proteins in E. coil AMINO ACID POSITION^(a) NET INDUCTIONEXPRESSION NAME (SIZE) CHARGE^(b) MEDIA^(c) TIME LEVEL^(d) MB-A  32-101+8.94 SB 1 hr 1-2 mg/L (70aa) 10 min MB2-B  95-206 +10.18 LB 8 hrs 3mg/L (112aa) MB2-C 200-316 +2.58 LB 4 hrs 4 mg/L (117aa) MB2-F^(e) 32-316 +21.91 SB 4 hrs 2 mg/L (A-B-C) (285aa) MB2-D 355-546 −0.72 SB1-6 hrs 0.1-0.2 mg/L (192aa) MB2-E 538-773 +8.23 SB 1-2 hrs 0.1-0.2 mg/L(236aa) MB2-FA 764-945 −17.51 SB 3-6 hrs 2 mg/L (182aa) MB2-IF21337-1606 +5.26 LB or 1 hr 0.5-1.0 mg/L (270aa) SB

[0090] Immunoblot analyses to determine the antigenic regions of MB2.Purified GST-MB2 recombinant proteins were used for immunoblot analysesto determine the antigenic regions of the molecule. The results of theimmunoblot analyses obtained with the serum from a protected volunteer(#5) showed that it contains mostly antibodies against the B domain(FIG. 6B). Minimal or no antibody reactivity was observed against therecombinant fragments of the A or G domains. Moreover, the antigenicproperty of MB2 is limited to the central region of the B domain, withantibody reacting strongly against the non-repeat-containing MB2-Bpeptide, and some antibody reactivity observed against therepeat-containing MB2-C peptide.

[0091] For comparison, the antigenicity of MB2 also was analyzed usingthe serum from an individual exposed naturally to P. falciparum (KU162,Table 2). As shown in FIG. 6B, as with the volunteer serum, the endemicserum contains no antibodies against the MB2-IF2 peptide from the Gdomain. However, in addition to antibodies against the B domain, theendemic serum also contains antibodies against the A domain.Furthermore, within the B domain, in contrast to serum from theprotected volunteer #5, the endemic serum contained antibodies thatstrongly react against the repeat-containing peptide, MB2-C. TABLE 2Individual antibody responses to MB2 recombinant peptides of the Bdomain Serum Source: MB2-A^(a) MB2-B^(a) MB2-C^(a) Volunteer serum:Volunteer #1 −^(b) ++^(b) −/+^(b) (protected) Volunteer #3 − ++ −/+(protected) Volunteer #5 − ++ −/+ (protected) Volunteer #7 − ++ −/+(protected) WRAIR #1 − ++ −/+ (protected) WRAIR #4 (not − − − protected)WRAIR #5 (not − − − protected) WRAIR #6 (not − − − protected) Kenyanserum: Smear negative (adults) ^(c)KU 036 − ++ + KU 071 − ++ +++ KU 081− + ++ KU 069 − + ++ KU 079 − −/+ ++ KU 083 − −/+ +++ KU 163 − ++ −/+ KU202 − − + KU 205 − −/+ −/+ Asymptomatic (adults) KU 118 − − −Asymptomatic (children) KU 044 − + − KU 048 − − + KU 076 − − −/+ KU 001− ++ ++ KU 049 − − − KU 025 − − − Mildly symptomatic (adults) KU 064 − −− KU 070 − + − KU 157 − + +++ KU 158 − + ++ KU 165 − − − KU 172 − −/+ ++KU 199 − + −/+ KU 203 − − −/+ KU 207 − − + KU 234 − + −/+ Mildlysymptomatic (children) KU 162 − + +++ KU 062 − − − KU 161 − − −/+ KU 041− − −/+ Severely symptomatic (adults) KU 037 − ++ −/+ KU 072 − −/+ −/+KU 075 − + ++ KU 080 − −/+ + KU 088 − − − KU 145 − + ++ KU 155 − − ++ KU174 − + + KU 183 − −/+ ++ Severely symptomatic (children) KU 067 − − +KU 084 − ++ +

[0092] Because the immunoblot analysis showed that the protectedvolunteer serum recognized principally the two regions in the B domain,and these regions were recognized differently between the volunteerserum and the endemic serum, it was important to determine if thisdifferential antibody recognition correlated with the different immunityobserved between protected volunteers and people living inmalaria-endemic regions. Thus, the immunoblot analysis was expanded toinclude serum obtained from 40 individuals living in Kenya withdifferent clinical status from blood-smear negative to severelysymptomatic. In addition, serum samples from seven other irradiatedsporozoite-immunized volunteers, of which four of seven acquired sterileimmunity following being immunized with irradiated sporozoites, wereincluded in the analysis.

[0093] Results of the expanded immunoblot analyses are shown in Table 2.A total of 83% (34/41) of naturally exposed individuals have anti-MB2antibodies. Of these, the analysis showed more individuals hadantibodies against MB2-C than MB2-B, 94% vs. 74%, respectively, and theintensity of the antibody reaction against the MB2-C region was almostalways stronger than the reaction against the non-repeat, MB2-B region.Minimal or no antibody reaction was detected against the amino-terminalMB2-A region. No obvious association between the anti-MB2 antibodyresponse and clinical immunity in the serum donors was observed.

[0094] In comparison, the immunoblot result obtained with sera ofirradiated-sporozoite immunized volunteers showed a 100% associationbetween the anti-MB2 antibody response and the immune status of thevolunteers (Table 2). All four additional protected volunteers producedanti-MB2 antibodies that are directed preferentially against thenon-repeat MB2-B region, a result similar to that obtained with serum ofthe initial single volunteer. In contrast, none of the three unprotectedvolunteers produced an antibody response against MB2. Anti-CS antibodieswere detected in all volunteer sera by ELISA (data not shown).

[0095] Polymorphism assessment of the antigenic region of the MB2 basicdomain. Because sequence variation of MB2 could contribute to thedifferent antibody recognition response observed between sera of thevolunteers and endemic persons, the antigenic regions in the B domain ofMB2 were evaluated for potential amino acid polymorphisms.

[0096] The nucleotide sequence of the MB2 gene was determined for fieldisolates obtained from different malaria-endemic regions of the world.It was expected that samples derived from non-overlapping locales wouldprovide the greatest opportunity to detect sequence variation in MB2.Only the nucleotide sequence encoding the first 317 amino acids of MB2was examined, as it is the region that contains B-cell epitopes thatwere recognized differentially in the immunoblot analysis.

[0097] The amino acid sequence alignment of the antigenic region of MB2from four laboratory strains and eleven field isolates collected fromIndia (3), Venezuela (4), Thailand (3) and Papua New Guinea (1) showthat the only variation observed is in the number of repeat units; twoout of eleven isolates (Ven-IS9 and PNG Muz37) have 7 versus 6 repeats(FIG. 7). There are no other polymorphisms detected outside the repeatregion, indicating that the amino acid sequence of the antigenic regionof MB2 is conserved absolutely among the laboratory strains and fieldisolates examined. The conservation of the antigenic region of the MB2gene resulted from the conservation of the nucleic acid sequence (datanot shown). No silent nucleotide polymorphisms were observed in any ofthe samples.

[0098] In vitro inhibition of sporozoite invasion assay. Because IEMstudies indicate that MB2 is located on the membrane surface ofsporozoites, the effect of the different antibody recognition againstthe molecule on the ability of sporozoites to enter hepatocytes wasassessed. An in vitro inhibition of sporozoite invasion (ISI) assay wasused to study this effect.

[0099] Total purified IgG from sera obtained from rabbits immunizedagainst three different regions of MB2 (MB2-B, -C, and -FA) were used inISI assays. The results show that the IgG against thenon-repeat-containing MB2-B peptide has the most inhibitory effect, 57%(Table 3). In contrast, the IgG against the repeat-containing peptide,MB2-C, showed only 18% inhibition. For comparison, the IgG fractionagainst another repeat-containing but non-antigenic peptide, MB2-FA,showed 33% inhibition. TABLE 3 Evaluation of in vitro antiparasiticactivities (inhibition of sporozoite invasion) of antibodies againstdifferent regions of MB2 SPOROZOITES/ IgG WELL MEAN + SD^(a) %INHIBITION^(b) MB2-B PRE-IMMUNE 522, 514, 593 543 ± 35 0 MB2-B 205, 251,238 231 ± 19  57% MB2-C^(c) PRE-IMMUNE 216, 312, 240 256 ± 40 0 MB2-C252, 177, 198 209 ± 31  18% MB2-FA^(c) PRE-IMMUNE 472, 485, 438 465 ± 200 MB2-FA 263, 354, 306 307 ± 37  33% Mab NFS1  11, 9, 6  8 ± 2  97% MEMCTRL 280, 350, 325 318 ± 29 0

[0100] Discussion of the immunological characterization of MB2. Thenovel P. falciparum antigen MB2 is a multi-domain sporozoite surfaceprotein. Immunoblot analyses using serum of a volunteer protected by theirradiated sporozoite vaccine shows that only the basic (B) domain ofMB2 is recognized, while the acidic (A) and GTP-binding (G) domains arenot. In comparison, serum from a person living in an endemic areacontains antibodies against both the B and A domains. It is expectedthat P. falciparum-exposed individuals should not contain anti-MB2antibodies directed against the G domain since it is not detected by IEMand immunoblot analyses of sporozoites recovered from mosquito salivaryglands as well as from sporozoites that had invaded the hepatoma cellline, HepG2-A16.

[0101] The immunoblot analyses revealed that all tested sera fromirradiated-sporozoite immunized and protected volunteers containantibodies against MB2. In contrast, no anti-MB2 antibodies weredetected in all tested sera of irradiated-sporozoite immunized but notprotected volunteers. Furthermore, antibodies from the serum ofprotected volunteers recognized preferentially the non-repeat-containingMB2-B peptide, while the repeat-containing MB2-C peptide is recognizedpreferentially by the serum of persons living in malaria-endemic areas.

[0102] In addition, although the A domain also contains two amino acidrepeat regions, serum from a protected volunteer showed minimal antibodyreactivity against the A domain. In contrast, two regions of the Adomain, one of which contains amino acid repeats, were recognizedstrongly in an endemic serum.

[0103] These results indicate that there may be one or more B-cellepitopes encoded in the non-repeat region, peptide MB2-B, that are morerelevant to protective immunity than those encoded in the repeatregions. Some authors have hypothesized that B-cell epitopes encoded bythe repeat sequence of Plasmodium parasites provide a “smoke-screen”effect to act as a decoy mechanism, shielding other more functionallyimportant epitopes from being detected (Anders, R. F., et al. (1986)Antigenic repeat structures in proteins of Plasmodium falciparum. In:Synthetic peptides as antigens. R. Porter and J. Whelan, eds.Chichester: John Wiley & Sons, pp. 164; Schofield, L. (1990) Bull. WorldHealth Org. 68, Suppl. 66). A similar observation also was reported inthe trypomastigote surface antigen-1 (TSA-1) of Trypanosoma cruzi, whichcontains a repeat region at the carboxyl terminus (Wrightsman, R. A., etal. (1994) J. of Immunology 153, 3148). Animals immunized with eitherthe full-length or the repeat-included carboxyl portion of TSA-1 do notsurvive a T. cruzi challenge, and the full-length TSA-1 immunizedanimals only contain anti-TSA-1 antibodies directed against the repeatregion. In contrast, a majority of animals immunized with thenon-repeat, amino-terminal portion of TSA-1 survive the challenge.

[0104] The results of the inhibition of sporozoite invasion assaysuggest that antibodies directed against epitopes encoded in thenon-repeat region (MB2-B) possess more antiparasitic activities thanantibodies against epitopes encoded in the repeat-included region(MB2-C). The in vitro ISI assay showed that anti-MB2-B antibodies aremore effective than anti-MB2-C and anti-MB2-FA antibodies at blockingsporozoites from invading the hepatocyte. Although 40% of the parasitesare still able to enter hepatoma cells, it is not known how theirintra-hepatic development is affected because the sporozoites are notable to complete their liver-stage development in the hepatoma cell usedin the ISI assay. The change of the cellular location of MB2 from thesurface to the nucleus as the parasite lifecycle progresses from thesporozoite stage to the erythrocytic stage suggests that it may have afunction in the development of the parasite. Thus, it is possible thatanti-MB2 antibodies, although they fail to block invasion completely,may hinder severely the intra-hepatic development of the sporozoite sothat the total effect of anti-MB2 antibodies can completely preventblood-stage development.

[0105] An assay that would provide further support for measuringprotective antibodies is the inhibition of liver stage development assay(ILSDA) (Charoenvit, Y., et al. (1997) Infect. & Immun. 65, 3430). TheILSDA requires primary human hepatocytes to support the completedevelopment of the liver stage of P. falciparum sporozoites. Studiesutilizing antiMB2 sera in an ILSDA should be illuminating.

[0106] Although all five protected volunteers produced anti-MB2antibodies preferentially against the non-repeat MB2-B region, and theISI assay showed that anti-MB2-B antibodies are more effective thanantibodies against other regions at blocking sporozoite invasion ofhepatocytes, it is not known if anti-MB2-B antibodies are protective invivo and responsible for the sterile immunity acquired byirradiated-sporozoite immunized volunteers. The immunoblot analyses withendemic sera showed no correlation between anti-MB2 antibodies and theclinical status of the infected persons. There are individuals who areblood-smear negative (no evident parasitemia) who have antibodiesagainst the repeat-included MB2-C region, and individuals withparasitemia and disease symptoms who have antibodies against thenon-repeat MB2-B region.

[0107] It is not known how the clinical status of the infected peoplewas achieved at the time of blood collection. It is possible that theirclinical status results from additional factors, such as antimalarialdrug use, as well as immune mechanisms. In this case, it would not bemeaningful to compare the characteristics of their anti-MB2 antibodyresponse to that of the protected volunteers. Furthermore, clinicalimmunity was assessed by the presence or absence of parasitemia andclinical symptoms at a single point in time, rather than by prospectiveanalysis of time to infection or incidence of clinical disease.

[0108] In addition, it is not known how the B cells of protectedvolunteers were able to recognize preferentially the non-repeat MB2-Band avoid the repeat-included MB2-C and -D regions that most naturallyexposed persons in malaria-endemic areas recognize. It is possible thatthe antigenic composition of the irradiated sporozoite is different fromthe normal one. It is known that at the optimal radiation dosagerequired to induce sterile immunity, the weakened sporozoite is not ableto develop completely in the hepatocyte (Sigler, C. I., P. Leland, andM. R. Hollingdale (1984) Am. J. Trop. Med. Hyg. 33, 544). Because thestage-dependent cellular localization of MB2 is accompanied bydifferential proteolytic processing, it may be that MB2 was notprocessed properly by the irradiated parasite and is mis-directed ontothe surface of infected hepatocytes such that the MB2-B region is nowaccessible to B cells.

[0109] Alternatively, it is observed that protected volunteers receivedhundreds to more than a thousand bites of infected and irradiatedmosquitoes to acquire sterile immunity (Herrington, D., et al. (1991)Am. J. Trop. Med. Hyg. 5, 539-547). In contrast, in malarious countries,most individuals receive on the average less than 200 infective bitesper year (Hay, S. I., et al. (2000) Trans R Soc Trop Med Hyg 94, 113)and in the highland area of Kenya, where the endemic serum samples usedherein were collected, the number of infective bites is likely to bemuch lower than this (Khaemba, B., A. Mutani, and M. Bett (1994) EastAfr. Med. J. 71, 195). It is possible that, due to an unknown mechanism,the quantitative difference of inoculated sporozoites is responsible forthe observed difference in antibody recognition against MB2 betweenprotected volunteers and persons living in endemic countries.

[0110] The analysis of the primary structure of amplified MB2 DNAfragments from different isolates of P. falciparum showed that antigenicvariation is unlikely to be a factor contributing to the differentantibody response against MB2. Except for variation in the number ofrepeat units, the antigenic region in the B domain of MB2 is absolutelyconserved among laboratory strains and field isolates collected fromdifferent parts of the world. The amino acid sequence conservation mayreflect a functional constraint of the B domain as it is not onlyexposed on the surface of the sporozoite but also translocated into thenucleus of blood-stage parasites. In the in vitro ISI study, antibodiesto this conserved, antigenic region of MB2 inhibit sporozoite invasionof hepatocytes. If these antibodies play a role in protective immunityin vivo, the finding that the antigenic region of MB2 is highlyconserved suggests that it is a likely target for immune attack byantibodies as most, if not all, sporozoites would be recognized.

[0111] Studies have shown that immunity to malaria also is mediated, atleast partly, by cellular immune mechanisms (Hoffman, S. L., et al.(1996) Attacking the Infected Hepatocyte. In: Malaria VaccineDevelopment. S. L. Hoffman, ed. American Society for Microbiology,Washington, D.C., p. 35). In endemic areas, cytotoxic T lymphocytes(CTLs) from exposed individuals recognize epitopes in a number ofpre-erythrocytic antigens of P. falciparum, and indirect evidenceindicates that these CTLs may play a role in protective immunity (Aidoo,M., et al. (1995) Lancet 345, 1003; Aidoo, M., et al. (1997)International Immunology 9, 731; Aidoo, M., et al. (2000) Infection andImmunity 68, 227). Because MB2 was shown to be expressed in multipledevelopmental stages including the hepatic stage that can be recognizeby CTLs, it is important to obtain evidence that MB2 also is recognizedin P. falciparum-exposed individuals.

[0112] The Plasmodium parasite is genetically complex, and based on therecent sequencing projects (Gardner M. J., et al. (1999) Parasitologia41, 69) may have 5,000-6,000 genes. Its antigenic composition also isexpected to be complex. Thus, the challenge in designing an effectiverecombinant malaria vaccine is to define the immunogenic molecule ormolecules that are essential, and the methods to present them properlyto the immune system to induce the desired immune responses that protectthe volunteers experimentally immunized with the irradiated sporozoitevaccine. The MB2 protein possesses a number of molecular and immunogenicproperties that indicate it is a novel immunogen and may represent animportant immune subject for vaccine studies.

[0113] Nucleotide and Amino Acid Sequences. Sequences identified as SEQID Nos 1-9 disclosed in this invention are new. These sequencesrepresent nucleotides and amino acid sequences of P. falciparum antigen.They were prepared according to the methods described in Examples 1-3.

[0114] SEQ ID NO 1 is the complete open reading frame (ORF) of MB2,constructed from overlapping sequences of cDNA clones.

[0115] SEQ ID NO 2 is cDNA clone c3-1-18.

[0116] SEQ ID NO 3 is cDNA clone c18-4-23.

[0117] SEQ ID NO 4 is cDNA clone c3-4-29.

[0118] SEQ ID NO 5 is cDNA clone Spz-MB2.

[0119] SEQ ID NO 6 is genomic clone g2-6-8.

[0120] SEQ ID NO 7 is genomic clone g2-4-4#5.

[0121] SEQ ID NO 8 is genomic clone g6-2-2.

[0122] SEQ ID NO 9 is the amino acid sequence of MB2.

[0123] SEQ ID NO 10 is a cDNA clone from isolate NF54.

[0124] SEQ ID NO 11 is a cDNA clone from isolate Ven-IS9.

[0125] SEQ ID NO 12 is a cDNA clone from isolate PNG-Muz37.

[0126] The MB2 cDNA and genomic sequences have been deposited inGenBank, accession numbers AF378132-AF378138 and AF454665-AF454667,inclusive. These sequences are hereby incorporated by reference in theirentirety.

[0127] In addition to the amino acid sequence of the MB2 polypeptideshown in SEQ ID NO 9, the present invention is also directed to variantsand derivatives of this polypeptide.

[0128] As used herein, the term “variant” refers to polypeptides inwhich one or more amino acids have been replaced by different aminoacids, such that the resulting variant polypeptide is at least 75%homologous, and preferably at least 85% homologous, to the basicsequence as, for example, shown in SEQ ID NO 9. Homology is defined asthe percentage number of amino acids that are identical or constituteconservative substitutions. Conservative substitutions of amino acidsare well known in the art. Representative examples are set forth inTable 4. TABLE 4 Original Residue Conservative Substitution(s) Ala SerArg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn,Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile, Phe Met,Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

[0129] Variants of polypeptides according to SEQ ID NO 9 may begenerated by conventional techniques, including either random orsite-directed mutagenesis of DNA encoding SEQ ID NO 9 (for examples, SEQID Nos 1-8). The resultant DNA fragments are then cloned into suitableexpression hosts such as E. coli using conventional technology andclones that retain the desired activity are detected. The term “variant”also includes naturally occurring allelic variants.

[0130] By “derivative” is meant a polypeptide that has been derived fromthe basic sequence by modification, for example by conjugation orcomplexing with other chemical moieties or by post-translationalmodification techniques as would be understood in the art. Suchderivatives include amino acid deletions and/or additions topolypeptides according to SEQ ID NO 9 or variants thereof wherein saidderivatives retain activity eliciting an immune response. Otherderivatives contemplated by the invention include, but are not limitedto, modification to side chains, incorporation of unnatural amino acidsand/or their derivatives during peptide, polypeptide or proteinsynthesis and the use of crosslinking agents.

[0131] Polypeptides of the inventions may be prepared by any suitableprocedure known to those of skill in the art. Recombinant polypeptidesof the invention may be produced by culturing a host cell transformedwith an expression vector containing nucleic acid encoding apolypeptide, fragment, variant or derivative according to the invention.Recombinant protein may be conveniently prepared by a person skilled inthe art using standard protocols as, for example, described in Sambrook,et al., MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring HarborPress, 1989), incorporated herein by reference, in particular Sections16 and 17; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (JohnWiley & Sons, Inc. 1994-1998), incorporated herein by reference, inparticular Chapters 10 and 16; and Coligan et al., CURRENT PROTOCOLS INPROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997) which isincorporated by reference herein, in particular Chapters 1, 5 and 6.Examples of vectors suitable for expression of recombinant proteininclude pGEX, pET-9d, pTrxFus or baculovirus (available fromInvitrogen). A number of other vectors are available for the productionof protein from both full length and partial cDNA and genomic clones,producing both fused or non-fused protein products, depending on thevector used. The resulting proteins are frequently immunologically andfunctionally similar to the corresponding endogenous proteins.

[0132] The obtained polypeptide is purified by methods known in the artor described in Examples. The degree of purification varies depending onthe use of the polypeptide. For use in eliciting polyclonal antibodies,the degree of purity may not need to be very high. However, as in somecases impurities may cause adverse reactions, purity of 90-95% istypically preferred and in some instances even required. For thepreparation of a pharmaceutical composition, however, the degree ofpurity must be high, as is known in the art.

[0133] Antibodies. The invention also contemplates polyclonal andmonoclonal antibodies against the aforementioned polypeptides,fragments, variants and derivatives. Methods of producing polyclonalantibodies are well known to those skilled in the art. Exemplaryprotocols which may be used are described for example in Coligan et al.,CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons, Inc, 1991) which isincorporated herein by reference, and Ausubel et al., (1994-1998,supra), in particular Section III of Chapter 11.

[0134] Alternatively, monoclonal antibodies may be produced using thestandard method as, for example, described in an article by Kohler andMilstein (1975, Nature 256, 495-497) which is herein incorporated byreference. Anti-P. falciparum polyclonal antibodies recognizing thecloned polypeptide are preferred over a monoclonal antibody (MAb)because they recognize multiple epitopes on the target polypeptide.

[0135] This invention further contemplates reagents such as recombinantsingle-chain or other antibody derivatives, including antibodylibraries, prepared using established procedures from mRNAs and/or cDNAsfrom hybridoma lines expressing antiMB2 antibodies. These reagents alsoinclude, but are not limited to, spleens, isolated spleen cells andmRNAs isolated from mice immunized with all or any portion of natural orsynthetic MB2 peptides.

[0136] According to the method of the current invention, large amountsof recombinant MB2, or derivative, variants or fragments thereof, areproduced by scale up processes in commercial plants which enablesproduction of a corresponding large quantity of polyclonal antibodiesand/or of immunogen for active immunization. The antibodies torecombinant expressed protein can also be produced according to theinvention using the standard method available for production of theantibodies to native protein.

[0137] The antibodies of the invention may be used for affinitychromatography in isolating natural or recombinant P. falciparumpolypeptides. The antibodies can also be used to screen expressionlibraries for variant polypeptides of the invention. The antibodies ofthe invention can also be used to detect P. falciparum infection.

[0138] Detection of P. falciparum. The presence or absence of P.falciparum in a patient may determined by isolating a body specimen,such as blood or other bodily fluid, from a patient, mixing an antibodyor antibody fragment described above with the biological sample to forma mixture, and detecting a complex of specifically bound antibody orbound fragment in the mixture which indicates the presence of P.falciparum in the sample.

[0139] Any suitable technique for determining formation of the complexmay be used. For example, immunoassays, such as radioimmunoassays(RIAs), enzyme-linked immunosorbent assays (ELISAs) andimmunochromatographic techniques (ICTs) are well known to those of skillin the art. For example, see “CURRENT PROTOCOLS IN IMMUNOLOGY” (1994,supra) which discloses a variety of immunoassays that may be used inaccordance with the present invention.

[0140] Examples of body specimens are stools and other liquid or solidbody output or tissue samples obtained from a subject. Examples of bodyfluids are blood, serum, saliva, urine, and the like. Methods for thepreparation of the body substance and the body fluid are standard in theart and are described, for example in Manual of Clinical Microbiology,Chapter 8, “Collection, Handling and Processing of Specimens”, 4thedition, Eds, Lennette, E. H., Balows, A., Hausler, W. J. and Shadorny,A. J., American Society for Microbiology, (1986)).

[0141] Diagnosis and detection methods also comprise contacting the DNAand RNA of body fluid, tissue, specimen and/or environmental sample withDNA and RNA of the invention or fragments thereof and the amplificationof this specific interaction via PCR, branched chain nucleic acidtechnology and other amplification technologies such that the presenceof P. falciparum DNA and/or RNA in the bodily fluid, tissue, specimen orenvironmental sample may be detected.

[0142] Agents suitable for immunodiagnostic use are proteins comprisingepitopes of P. falciparum that are recognized by intact B and/or Tcells. These proteins are produced as described above, purified and usedto detect or characterize anti-P. falciparum antibody in the bodysubstances of populations at risk of prior or current P. falciparuminfection. In addition, antibodies to such proteins are obtained byimmunizing animals, such as cows, rabbits or goats, or birds with thevaccine combined with an adjuvant.

[0143] Pharmaceutical compositions. Another aspect of the invention isthe use of the MB2 polypeptide, fragment, variant or derivative of theinvention (collectively, “immunogenic agents”) as actives in apharmaceutical composition for protecting patients against infection byP. falciparum. Suitably, the pharmaceutical composition comprises apharmaceutically acceptable carrier.

[0144] A “pharmaceutically-acceptable carrier” is a solid or liquidfiller, diluent or encapsulating substance that may be safely used insystemic administration. Depending upon the particular route ofadministration, a variety of pharmaceutically acceptable carriers, wellknown in the art may be used. These carriers may be selected from agroup including sugars, starches, cellulose and its derivatives, malt,gelatine, talc, calcium sulfate, vegetable oils, synthetic oils,polyols, alginic acid, phosphate buffered solutions, emulsifiers,isotonic saline, and pyrogen-free water.

[0145] The pharmaceutical composition of the invention may beadministered by any of the conventional routes of administration,including oral, rectal, parenteral, sublingual, buccal, intravenous,intra-articular, intramuscular, intra-dermal, subcutaneous,inhalational, intraocular, intraperitoneal, intracerebroventricular,transdermal and the like. Also, the pharmaceutical composition of theinvention may be in any of several conventional dosage forms, including,but not limited to, tablets, dispersions, suspensions, injections,solutions, capsules, suppositories, aerosols, and transdermal patches.

[0146] The above compositions may be used as therapeutic or prophylacticvaccines. Accordingly, the invention extends to the production ofvaccines containing as actives one or more of the immunogenic agents ofthe invention. Any suitable procedure is contemplated for producing suchvaccines. Exemplary procedures include, for example, those described inNEW GENERATION VACCINES (1997, Levine et al., Marcel Dekker, Inc. NewYork, Basel Hong Kong) and U.S. Pat. No. 6,254,869, both of which areincorporated herein by reference.

[0147] Thus, the invention describes vaccines able to provide active Bcell-immunity and potentially T cell immunity against malaria. Typicalintramuscular immunization schedules are as follows. The immunogenicagents, plus equal volume complete pharmaceutically acceptable adjuvantsand excipients is used at the beginning of immunization. Immunogenicagents plus equal volume incomplete adjuvant is used at week 2.Immunogenic agents plus equal volume incomplete adjuvant is used at week4.

[0148] DNA or RNA vaccines. DNA or RNA vaccines have been described inScience, 259:1745 (1993), hereby incorporated by reference in itsentirety. Briefly, nucleic acid vectors containing DNA or RNA encodingP. falciparum immunogenic agent(s) are injected, preferablyintramuscularly, into the host, where the vector is expressed toproduced antigen. The antigen elicits immune responses in the form ofspecific anti-P. falciparum antigen antibody or cell mediated immuneevents. In this way, the host receives DNA or RNA and provides his orher own humoral immunity and/or cell mediated responses.

[0149] Immunotherapy and prophylaxis. A method for immunotherapeutictreatment, retardation, or inhibition of P. falciparum infectioncomprises administering to a subject in need of such treatment an amountof an anti-P. falciparum polyclonal or monoclonal antibody preparedaccording to the invention, effective to provide immunity against theinvasion of P. falciparum or effective to inhibit the existing P.falciparum infection.

[0150] A method of prophylaxis of P. falciparum infection comprisesadministering to a subject in need of such treatment a vaccine, asdescribed above, comprising the protein or recombinant polypeptide ofthis invention capable of endogenous development of inhibitory amount ofanti-P. falciparum antibodies.

[0151] Typical immunization is achieved by inoculation of the animal,bird or human host with the antigen protein combined with adjuvant.

[0152] For passive immunotherapy when used to passively immunize P.falciparum infected hosts, the polypeptide is first combined withappropriate adjuvants and used for the immunization of cows or otherdonor animals to produce antibodies which may be administered topatients with malaria. Monoclonal antibodies produced in animals, inhumans “humanized” from animal sources and produced through chimerictechniques and other derivative techniques may be used for passiveimmunotherapy.

[0153] When in a therapeutic composition, the antigen protein iscombined with appropriate adjuvants and used for the immunization ofpatients who are at risk for malaria either at the time of immunizationof in the future.

[0154] Qualitative and quantitative detection of P. falciparum:formulations and kits. Formulations suitable for the administration ofpolypeptides and antibodies such as those described herein are known inthe art. Typically, other components stimulatory of immune response aswell as fillers, coloring, and the like may be added, such aspharmaceutically acceptable excipient, additives and adjuvants.

[0155] For qualitative and quantitative determination of the presence ofthe P. falciparum infection and environmental contamination, a kit forthe diagnosis/detection of P. falciparum is used. The kit comprises thepolyclonal antibody or antigen of this invention and a means fordetecting the completing of the antibody with antigen.

[0156] Another such kit comprises DNA/RNA of the invention for use indetecting complementary DNA/RNA of P. falciparum MB2. Another such kitcomprises PCR primers for amplification of MB2 sequences and a method ofidentifying them.

[0157] The kit is utilized for the detection of endogenousantibodies/antigens/DNA/RNA produced by a subject that is afflicted withmalaria. Even at the early stages where the parasite is commencinginvasion of a subject's cells, some amount of the P. falciparum antigenor the specific antibody may be detected in serum. The kit is alsoutilized for the detection of antigens/DNA/RNA present in theenvironmental samples, including, for example, testing of mosquitoes forthe presence of Plasmodium.

[0158] The kit detects either the antigen with the polyclonal antibodiesor the presence of the anti-P. falciparum antibody with the antigen. Thecomplexing immunoreaction is detected by staining, radiography,immunoprecipitation or by any other means used in the art and suitablefor these purposes.

[0159] In addition to the above, the kits may also comprise a controlcompounds, anti-antibodies, protein A/G, and the like, suitable forconducting the different assays referred to above.

[0160] Having generally described the invention, the same will be morereadily understood through reference to the following examples, whichare provided by way of illustration, and are not intended to be limitingof the present invention.

Example 1

[0161] Library Construction. A CS-depleted sporozoite cDNA library wasconstructed from a P. falciparum salivary gland sporozoite cDNA library(strain NF54; Fidock, D. A., et al. (2000) Exp. Parasitol. 95, 220-225)using a hydroxyapatite column-based subtractive hybridization technique(Usui, H., et al. (1994) J. Neurosci. 14, 4915-4926). To prepare thetarget cDNA sense-strands, DNA from the unsubtracted library waslinearized with NotI and used as a template to transcribe antisensecRNAs with T7 RNA polymerase (Megascript, Ambion). Template DNA wasremoved by DNase treatment and the antisense cRNA strands were used togenerate cDNA sense strands in a reaction using SuperScript (LifeTechnologies, Inc.) reverse transcriptase. To prepare the driver cRNA, aCS clone, G89 (McCutchan, T. F., et al. (1984) Science 225, 625-628),was linearized with NotI. Digestion products were used to generateantisense CS cRNA with T7 RNA polymerase (Megascript, Ambion).

[0162] The target cDNA sense strands were allowed to reassociate with a50-fold excess of the driver cRNA antisense strands. The reassociationmix was loaded onto a hydroxyapatite column and nonduplex,single-stranded target cDNA was separated from duplex cDNA/cRNA byelution with a high molarity phosphate buffer. Primers specific for theUniZap λ phage vector (Stratagene) were used to amplify the subtractedcDNA, and the amplification products were subcloned into the phage armsof the UniZap vector and packaged.

Example 2

[0163] Library Screening. Phage were plated and lifted ontonitrocellulose membranes that were soaked in 10 mMisopropylthio-β-D-galactoside and air-dried prior to use. Membranes wereincubated in the serum of human volunteer 5 at a 1:100 dilution, andhorseradish peroxidase-conjugated anti-human IgG+IgM were used to detectpositive antibody reactions by the ECL system (Amersham PharmaciaBiotech). Positive phage were screened a second time to isolate singlephage clones. Additional cDNA and genomic clones (strain ITO) wererecovered using MB2-derived ³²P-labeled probes and standardlibrary-screening techniques (Sambrook, J., Fritsch, E. F., andManiatis, T. (1989) Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.).

Example 3

[0164] DNA Sequencing of MB2. The primary nucleotide sequences of allclones were determined by the dideoxynucleotide chain termination method(Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad.Sci. U.S. A. 74, 5463-5467) using a ³³P nucleotide terminator kit(Amersham Pharmacia Biotech). Specific oligonucleotide primers forsequencing were made by Heligen Laboratories (Huntington Beach, Calif.).Contiguity of clones was verified by gene amplification of genomic DNAusing the following primers: a, 5′-GGTGATGACATTGAAGATATGAATG-3′; b, 5′CAATAGAATAGATATAATCACC; c, 5′-CTGGGTCATCATATGGAAAAGTG-3′; and d,5′-CAATACACCCTGCAACCTTTCC-3′.

Example 4

[0165] Southern and Northern Analyses. P. falciparum genomic DNA wasisolated using a phenol/chloroform-based procedure (Sambrook et al.,supra) from blood-stage parasites (strain FCR3) cultured in vitro. TheDNA was digested with various restriction endonucleases, and Southernblots were prepared as described (Id.). The probe for Southern blotanalyses was prepared by labeling the sporozoite cDNA clone, spz-MB2,with radioactive [³²P]ATP using the Megaprime DNA system (AmershamPharmacia Biotech). Total RNA was isolated from blood-stage parasites ofthe same strain cultured in vitro using the TRIZOL® reagent (LifeTechnologies, Inc.). Total RNA (15-20 μg) was electrophoresed andNorthern blots were prepared as described (Id.). Two ³²P-labeled probesconsisting of nucleotides 1-580 and 2393-2836 of the coding sequence ofMB2 were used separately on filters to which RNA from blood-stageparasites had been transferred.

Example 5

[0166] Recombinant Protein Expression and Purification Fragments of theMB2 open reading frame (ORF) were expressed in bacteria as GST-MB2-6×His fusion proteins in the dual-affinity pAK1-6H expression vector(Stratmann, T., et al. (1997) Protein Expression Purif. 11, 72-78). NcoIand SmaI cloning sites were created for each insert by amplifying NF54strain genomic DNA. The names, positions, and nucleotide sequences ofoligonucleotide primers used to amplify, clone, and express GST-MB2recombinant proteins are listed in Table 6. TABLE 6 Names, positions,and sequences of primers used to clone and express GST-fusion proteinsrepresenting various regions of the MB2 open reading frame.5′ primer-Nco I 3′ primer-Sma I GST- (5′ to 3′) + (5′ to 3′) + Aminofusion strand strand acid^(a) MB2-A ^(b)(98)GATGCCATGGGTG(305)GATCCCGGGGAGC 32- TTAATAGATGTTTTATC ATATTCTATTATATTCA 101 MB2-B(286)GATGCCATGGAAT (620)GATCCCGGGTTTT 95- ATAATAGAATATGCTCATATTATTAGAAGAATCA 206 MB2-C^(C) (602)GATGCCATGGATT (953)ATGCATCCCCGGG200- CTTCTAATAATAAAAAT TCATTTTTTATTTGAAGA 316 ATTCTC MB2-F^(d)(98)GATGCCATGGGTGT (953)ATGCATCCCCGGG 32- TAATAGATGTTTTATCTCTTTTTTATTTGAAGAA 316 TTCTC MB2-D^(c) (1066)GATGCCATGGCA(1640)GATCCCGGGGAT 355- TCTACATTAGATGAAACA GTACTATAATCATTATTT 546 GGMB2-E (1616)GATGCCATGGAT (2321)GATCCCGGGCTT 538- CCAAATAATGATTATAGTGAATTATATTCTTTATTT 773 ACA TCGTG MB2- (2294)GTATGCCATGGT(2837)GATCCCGGGTCA 764- FA^(c) CCACGAAAATAAAGAATA TCGAGCGATTCATTTTGG 945TAATTCAAG TC MB2- (4009)GATGCCATGGAT (4823)GATCCCGGGTAC 1337- IF2GGTAATAGAACAAATAAT GCTTCGATTATATCGTTT 1606 GAC GGCTC

[0167] The amplification products were digested with NcoI and SmaI andligated into pAK1-6H. The ligation mixture was used to transformEscherichia coli DH10B, and transformants were selected. Bacterial cellswere grown at 37° C. in SuperBroth (Life Technologies, Inc.) to anA₆₀₀=0.6 and induced in 1 mM final concentration ofisopropylthio-β-D-galactoside for 3 to 4 hours to express recombinantproteins. Purification of recombinant proteins was done using theProBond resin (Invitrogen) modified by the inclusion of imidazole at 85mM final concentration in the washing buffer. Eluted fractions wereanalyzed by SDS-PAGE and immunoblotting for the presence of recombinantproteins using anti-GST antibodies.

Example 6

[0168] Rabbit Immunization. Purified recombinant protein (400 μg) wasinjected subcutaneously into a rabbit four times at 2-week intervals.Ten days following the last injection, high titer sera were obtainedfrom the rabbit. The sera were depleted of anti-GST antibodies bychromatography on GST-bound nickel columns.

Example 7

[0169] Immunoelectron Microscopy. P. falciparum parasites andparasite-infected cells or tissues were fixed for 30 min at 4° C. with1% formaldehyde, 0.1% glutaraldehyde in a 0.1 M phosphate buffer, pH7.4. Fixed samples were washed, dehydrated and embedded in LR Whiteresin (Polysciences, Inc.). Thin sections (70-80 nm) were blocked in aphosphate buffer containing 5% w/v nonfat dry milk and 0.01% v/v Tween20 (Aikawa, M., and Atkinson, C. T. (1990) Adv. Parasitol. 29, 151-214).

[0170] Grids were incubated at 4° C. overnight in solutions containingvariable concentrations of rabbit antiserum reactive to domain-specificrecombinant proteins diluted in the blocking buffer. Preimmune sera wereused as negative controls. After washing, grids were incubated for 1 hin 15 nm gold-conjugated goat anti-rabbit IgG (Amersham PharmaciaBiotech) diluted 1:40 in phosphate buffer containing 1% bovine serumalbumin and 0.01% Tween 20. Following the 1 h incubation, grids wererinsed with phosphate buffer containing 1% bovine serum albumin and0.01% Tween 20 and fixed with glutaraldehyde to stabilize the goldparticles. Samples were stained with uranyl acetate and lead citrate andexamined by electron microscopy.

Example 8

[0171] Immunoblot Analysis (Rabbit). Protein extracts from parasiteswere prepared by boiling them in sample buffer for 10 min (Laemmli, U.K. (1970) Nature 227, 680-685). For the sporozoite stage, parasites wereisolated from dissected salivary glands of infected mosquitoes. For theasexual blood stages, parasites were obtained from a saponin lysis ofinfected red blood cells grown in culture (Hyde, J. E., and Read, M.(1993) Methods Mol. Biol. 21, 133-143).

[0172] Protein extracts were fractionated on 8% SDS-polyacrylamide gelelectrophoresis and transferred onto nitrocellulose membranes. Themembranes were incubated in rabbit antiserum diluted 1:500 for 1 hour.Horseradish peroxidase-conjugated anti-rabbit IgG was used to detectpositive signals using the ECL kit (Amersham Pharmacia Biotech).Preimmune sera and lysates from uninfected human red blood cells wereused as negative controls.

Example 9

[0173] Human Sera. Samples of human serum from individuals exposednaturally to malaria were collected from donors living in Kenya, amalaria-endemic country in East Africa. All donors were exposedpreviously to P. falciparum infected mosquitoes as evidenced by thepresence of anti-CS and anti-TRAP antibodies in their sera.

[0174] The clinical status of the serum donors at the time of bloodcollection was known and was defined in four categories; blood-smearnegative and asymptomatic, blood-smear positive and asymptomatic,blood-smear positive and mildly symptomatic, and blood-smear positiveand severely symptomatic. Blood-smear negative persons had a lack ofevident peripheral blood P. falciparum parasitemia. Blood-smear positivepersons had parasites in their red blood cells and were divided into thefollowing categories: severely symptomatic persons had the physicalsigns of either fever or pallor plus three or more of the followingsymptoms: antecedent fever, joint pains, headache, chills vomiting, orsevere fatigue; mildly symptomatic persons had no fever or pallor, buthad two or more of the above symptoms; asymptomatic persons had no feveror pallor, and had none of the above symptoms. Informed consent wasobtained from all individuals or their guardians, as describedpreviously (John, C. C., et al. (2000) Infect. Immun. 68, 5198).

[0175] Samples of serum from eight volunteers experimentally immunizedby the bites of irradiated, infected mosquitoes were obtained from A.Kang, formerly at The Scripps Research Institute (La Jolla, Calif.);from W. O. Rogers at the US Naval Medical Research Center (Rockville,Md.); and from U. Krzych at the Walter Reed Army Institute of Research(Washington D.C.). Five of the eight volunteers were resistant to thechallenge of the bites of infected, nonirradiated mosquitoes, and theother three were not (W. Rogers and U. Krzych, personal communication).

Example 10

[0176] Immunoblot Analyses (Human). Purified GST-MB2 recombinantproteins (˜50-100 ng) were resolved by SDS-PAGE in a 12% polyacrylamidegel and the proteins were blotted onto a nitrocellulose membrane. Themembrane was reacted with the human serum at a 1:100 dilution inTris-buffered saline (TBS) plus 0.05% Tween 20 for two hours at roomtemperature. HRP-conjugated anti-human IgG at 1:80,000 dilution(CalBiochem) was used to detect positive antibody reactions by the ECLsystem (Amersham). Rabbit anti-GST antibodies were used as a positivecontrol and normal human serum was used as a negative control.

Example 11

[0177] Amplification and Sequence of MB2 Genes from Diverse ParasitePopulations. P. falciparum genomic DNA from laboratory-maintainedstrains and field isolates (kindly provided by Dr. A. Lal, Centers forDisease Control and Prevention, Atlanta, Ga.) was extracted and purifiedfrom infected red blood cells. The nucleotides encoding the antigenicregion of the B domain of MB2 (amino acids 1-317) were amplified fromgenomic DNA by the polymerase chain reaction (PCR) using the followingprimers: 5′-ATGTTTCTAATATGGCGTTTG-3′ and 5′-TCATTTTTATTTGAAGAATT-3′.Conditions for amplification included an initial DNA denaturation of 94°C. for 2 min, followed by 30 cycles at 94° C. for 20 s, 55° C. for 20 s,and 60° C. for 1 min. Genomic DNA (200 ng) from each lab strain was usedas template in the reaction. The concentrations of genomic DNAs of fieldisolates used as template in the amplification reaction were not knowndue to the limited quantities of material in each of the samples.

[0178] The amplification products were cloned directly into a TA cloningvector using the TOPO-PCR cloning kit (Invitrogen) Plasmid DNAs wereprepared from bacterial cultures and both strands of the genes weresequenced. Cloning and sequencing were repeated a second time onamplification products obtained independently to ensure reproducibilityof the sequence data. Alignment of the sequences was performed by theClustal method using the Megalign program from the Lasergene computersoftware. Novel sequences have been deposited in GenBank with accessionnumbers AF454665-AF454667.

Example 12

[0179] Rabbit Total IgG Purification. Rabbit immunization procedures toobtain polyclonal antibodies against MB2 recombinant proteins wereperformed as described in Example 6. For IgG purification, theIMMUNOPURE® IgG Protein A Purification Kit was used, and thepurification procedure was followed essentially as described by themanufacturer (Pierce, Ill.).

Example 13

[0180] In Vitro Inhibition of Sporozoite Invasion (ISI) Assay. ISIassays were performed to assess the inhibitory effects of antibodies onthe invasion of cultured liver cells by sporozoites (Hollingdale, M. R.,et al. (1984) Journal of Immunology 132, 909; Charoenvit, Y., et al.(1997) Infect. & Immun. 65, 3430). A human hepatoma cell line,HepG2-A16, (Schwartz, A. L., et al. (1981) J. of Biol. Chem. 256, 8878)was used for the assay.

[0181] Approximately 50,000 HepG2-A16 cells were seeded on eight-chamberplastic Lab-Tek slides (Miles Research) in supplemented minimalessential medium as described (Charoenvit, Y., supra). The purifiedantibodies from Example 12 were added to a final concentration of 100mg/ml, and HepG2-A16 cells were infected with 25,000 P. falciparum NF54sporozoites.

[0182] Slides were incubated at 37° C. in 5% CO₂ for 3 hours, washedtwice with phosphate-buffered saline (PBS), fixed with cold methanol,and rinsed twice again with PBS. Sporozoites that had invaded hepatomacells were visualized by phase-contrast light microscopy usingimmunohistochemical staining with a mouse monoclonal antibody (NFS1)against P. falciparum CS protein. Slides were reacted with NFS1 (10μg/ml) for 30 min at room temperature, followed by incubation withperoxidase-conjugated goat anti-mouse IgG for 30 min at roomtemperature.

[0183] All cultures for ISI assays were done in triplicate withpreimmune IgG used as the negative control and the anti-CS monoclonalantibody NFS1 used as the positive control. The NFS1 antibody used forpositive control consistently produces a >90% inhibition.

[0184] All patents, patent applications, and other publicationsmentioned in this specification are incorporated herein in theirentireties by reference.

[0185] While this invention has been described in detail with referenceto a certain preferred embodiments, it should be appreciated that thepresent invention is not limited to those precise embodiments. Forexample, although one embodiment is described with reference to P.falciparum, homologous MB2 sequences have been detected in otherPlasmodium species, including P. yoelli, P. berghei, and P. gallinaceum;it is further anticipated that homologous sequence for MB2 will bedetected in P. vivex. Accordingly, this invention encompasses allPlasmodium species containing MB2 homologous sequence.

[0186] Rather, in view of the present disclosure which describes thecurrent best mode for practicing the invention, many modifications andvariations would present themselves to those of skill in the art withoutdeparting from the scope and spirit of this invention. The scope of theinvention is, therefore, indicated by the following claims rather thanby the foregoing description. All changes, modifications, and variationscoming within the meaning and range of equivalency of the claims are tobe considered within their scope.

1 12 1 5314 DNA Plasmodium falciparum 1 aaaaaaacaa agaaacaaaa aagagatgtgaataagttta tgatgtgaat aaatttatga 60 tgtgtataaa tttatgatgt gaataaatttatgatgtgaa taaatttatg atgtgaataa 120 atttatgatg tgtataaatt tatgatgtgaataaatttat gatgtgaata aatttatgat 180 gtgaataaat ttatgatgtg aataaatttatgatgtgtat aaatttatga tgtgtataaa 240 tttatgatgt gtataaattt atgatgtgtatatatttatg atgtgaataa atttatgatg 300 tgtataaatt tatgatgtga ataaatttatgatgtgtata aatttatgat gtgtatttat 360 ttttcttttt tttagtttta tataattatacatacatata tatatatata tatatttata 420 tatatataca aagggatgtt tctaatatggcgtttgtata aaaccttatt cttaatattg 480 tatattataa ttttacaaga atatgtatgtcattcttcta atcatataaa aagtgttaat 540 agatgtttta tcccctttat ttcagaatatatatataaac agaaaaacag aaaaaataat 600 atatcgtatg attctagtaa cagtcactacaatgataaaa taataccatg tggtaattat 660 aataaacaat taaaatataa aataaatttttgtcgtaatt tgtattttaa taataaaaat 720 gaatataata gaatatgctc aaggaacaaattaaattttc ataatataca aacagataat 780 acaatataca aaccaaaaaa gaaatattatgaagttggaa aagtaagaga aaaaataaaa 840 atgtataccc tttttaaaga tgacaaaataaacacactga aatgtaatga agaagaatct 900 gttacttcag aaacaataaa taagcatgattcaagtgtaa aagtaaaagg aagaagaaaa 960 aaaaatataa caagtaatga aaatattatgaatccaaata ataaaagtgt aagtagcatt 1020 aatacaaatc taagtgattc ttctaataataaaaatgata attccttaaa tagtaaaaaa 1080 aatgataata ccttaaatag taaaaaaaatgataatacct taaatagtaa aaaaaatgat 1140 aataccttaa atagtaaaaa taataacaattccttaaata gtaaaaataa taacaattcc 1200 ttaaatagta aaaagagtaa taattcttataatgacaaac atatagacca tataattcct 1260 gaaggcaaaa ataaaataaa caataatatagatgtaaaac acaatattaa taacaaatta 1320 aatgaaatta atgaagaacc ttatgaagatacacataata aagaagagaa ttcttcaaat 1380 aaaaatgata atgatgaaaa aagaaaagaagaaaataata atataacaag aagatatata 1440 aaaaatgacc aaatgtcata taataatataaatacaaatt ctaatgaata tgacaaaaat 1500 gcatctacat tagatgaaac atatataggtaaaacttttg aaggttatgt ttatagtgtt 1560 aataaaaatg ctgcgtgtat taaattaaaaaatattaata aatatggttt gttatttaaa 1620 aacaaagcaa atttaggtga tgacattgaagatatgaatg atttttttga aaaagatcaa 1680 ccggttcatg tgaaaatact tggtattaatacaaagaaga atatatttta tctaggaaat 1740 attataaaat ataatgaaaa tataaaattaagtaaaggag aatattcaaa gggattaata 1800 acaaaagtat gtgattccta ctgttttattaaagttttaa aaaatggaag cactggatat 1860 ttacataaat caaaattgtt ttgtatgaatgataaagaaa agaaaaataa tgataatcaa 1920 aattatgata atccaaataa tgataatccaaattatgata atcaaaatta tgataatcaa 1980 aattataata atccaaatta tgatcatccaaattatgata atcaaaatta tgataatcaa 2040 aattataata atccaaataa tgattatagtacatcacaat tatataacag tgataattta 2100 caaatggatt ttatatataa attacaatttacaaaaatat ttaatatatg ggatataata 2160 gatgttgaaa tattaggaac tccacaaaatgattataaat caaattatat attgacaata 2220 ccaagaggat ctaaaacatt taagaaaattttgaattatt tgaatgtttt aaaagaaaat 2280 gaagatatta ataatataca atataaaggtgattatatct attctattga taataataaa 2340 aatgacaata taatagattc agatataaataatcatcaca ttaataataa gaagaaaaag 2400 aagaatctat atgatataca aaataatatgaatcattctc cttttaataa gtttcataca 2460 gaagatgaat atttatttaa tgaccatgtccaagaaaatg ttcacacctt ttatgaaaaa 2520 aataaaaaat ataaaattac atatgataaagaaaataatc ataaaatgaa taaatcgtat 2580 tatttaaaaa aaaataaaga attaccttttaataataaat tcaaaaaaat cattaaaaat 2640 atttatgacc ttcctaacac tatttctttatctatgttat ctaaaacaat taaaatacca 2700 ttggcatcta taaaaaaata ttttatcatccacgaaaata aagaatataa ttcaagttat 2760 aaaattaatt cagaacagat aaaacgaatatgtcaacatt tcaaaataga ctgtaatgta 2820 gaacagagag atgataatgt ggttacaaaagtgaatggga caactaagga ttgtcaagaa 2880 aaagttttta aaaatgtgac acaggacagattgaaggaag gtgaacagga acgagttatt 2940 aaggtggaag caaagattaa aaatgatgaaatggttatgc aagagcaaaa agatactaag 3000 gaggagaagc acatggacgt tcaatttattgaagaaaaag atattaatgt acaacatatt 3060 aatgtacaag atatggatgt acaagatatggatgtacaag atattaatgt acaagatatg 3120 gatgtgcaaa atattaatgt acaagatattaatattcaag atatggatgt gcaaaatatt 3180 aataacagta taacacttaa caaatcgacaagttgtcaaa ccgatgaatc gcgagacgca 3240 ccggggggtg accaaaatga atcgctcgatgaaaaagatt cgatggaaaa aagtaaagaa 3300 aaaaaaaaaa aaaaagggaa aagtagaaaaaaaaataaag ataccaattt aacattaaaa 3360 agtgatagta ttcaaaaatc aaagaccaccctagacgata aaaaacgaaa tgtggtagtt 3420 acatttatag gacatattaa tcatggaaaaacgtctttat ttgattatat atgtaaaacc 3480 aatgaacaaa aaaaggagta tggacttataacacaaaata taagagcgtt taaagcaacc 3540 gtaaggaata attttacatt tactcttgtcgataccccag gacatgaagc atttatgcct 3600 atgagaagta gaggtgttaa aatatcagatttaagtattc ttgttatatc aggagatgaa 3660 ggaatacaag aacagactgt ggaatgtataaaattaataa aagaatttaa tattaaaatt 3720 attattgcaa taactaaagt agatattcctaatgttgatg tagatagaat aattaatgat 3780 ttgttatatc atgatataac aacagaattaaatggagggg aaatacaagt agttgaatgt 3840 tctatttata aagaagaaag tatagataaattattagatg ccatatattt agaatctgaa 3900 tttcttaatc tacaaaccaa tcctgataagaaacatgaac aggctcaagg tgttgtttta 3960 gattcttata tagataaaaa tggaattgtttctataaatt tgttacaaaa tggtgtatta 4020 aatataaatg atcattttta tactgggtcatcatatggaa aagtgaagat attaaaagat 4080 catttaaata aaaatattaa aagtgcatatccatcggatc ctattaaaat tattggatac 4140 aacaaaaatt ctgttcctgt agcaggtgacaaattttatg ttgttgaaaa tgaagcccta 4200 gccaaagaaa ttgcggaaca taataagaataaaatgttaa caatggaaat taataatttt 4260 acttatgatc agacaaatat gaacaggtataaagatttta taatatccag agaaaataaa 4320 attggaggtt cttcaggtat actgggagaaaataatttaa aaaatgacat tgatggtaat 4380 atgacaaggg atgataatat gacaagggatgataatatga caagtgatga taatatgaca 4440 agggatggta atagaacaaa taatgacaatattacaagtg atgataatat gtcaaatgat 4500 tatgataaaa taaaagaaac gaaaatgtatacaaataata aatcatttca aaaggatgat 4560 tttttaaaaa tacatttgaa taatacaaacgaaaatgtga ttaatatgga tccttcaaca 4620 catataggaa aaaatgaaat aaaaacaatatactcaaatt atattattaa atgtgataaa 4680 caaggaagta tagaagtttt gaaaaattgtatgcttaaat tacaaaaaga agatagtata 4740 tgtaaaataa aaaacaaaat tatatatgctgatataggta atgtaacatc aagtgatata 4800 aaatatgcta caagttttaa tgctacaataatagcttttg gtgttaaatt atcaaatgat 4860 attaaaggtt caaaaaattc aaaaggttcaaaaaatcata ataattatcc tattatatat 4920 tcaaatgtct tatatgaact tatagaaaatgtggaaaaag aaatggaaaa gaaattaagt 4980 aaaaaaccaa tgggtgaatt aaaaggaacagcacaaattt taaaagtttt taatatatcg 5040 aaacttggaa aggttgcagg gtgtattgttaaaaaaggta ctatctctat aaatagtaat 5100 attcgtattt taagaaatga taaagttatttatatgggaa aaatcatttc tattaaaatt 5160 gttaaggaag aaaaaacaca agtaacggaagctgatgaat gtggtatagg ttttgataat 5220 tttttggatt ttgagccaaa cgatataatcgaagcgtacg aaaattaaat atgaaaaaaa 5280 aaaaaaaaaa tgtaatgtaa aaaaaaaaaaaaaa 5314 2 1910 DNA Plasmodium falciparum 2 aaaaaaacaa agaaacaaaaaagagatgtg aataagttta tgatgtgaat aaatttatga 60 tgtgtataaa tttatgatgtgaataaattt atgatgtgaa taaatttatg atgtgaataa 120 atttatgatg tgtataaatttatgatgtga ataaatttat gatgtgaata aatttatgat 180 gtgaataaat ttatgatgtgaataaattta tgatgtgtat aaatttatga tgtgtataaa 240 tttatgatgt gtataaatttatgatgtgta tatatttatg atgtgaataa atttatgatg 300 tgtataaatt tatgatgtgaataaatttat gatgtgtata aatttatgat gtgtatttat 360 ttttcttttt tttagttttatataattata catacatata tatatatata tatatttata 420 tatatataca aagggatgtttctaatatgg cgtttgtata aaaccttatt cttaatattg 480 tatattataa ttttacaagaatatgtatgt cattcttcta atcatataaa aagtgttaat 540 agatgtttta tcccctttatttcagaatat atatataaac agaaaaacag aaaaaataat 600 atatcgtatg attctagtaacagtcactac aatgataaaa taataccatg tggtaattat 660 aataaacaat taaaatataaaataaatttt tgtcgtaatt tgtattttaa taataaaaat 720 gaatataata gaatatgctcaaggaacaaa ttaaattttc ataatataca aacagataat 780 acaatataca aaccaaaaaagaaatattat gaagttggaa aagtaagaga aaaaataaaa 840 atgtataccc tttttaaagatgacaaaata aacacactga aatgtaatga agaagaatct 900 gttacttcag aaacaataaataagcatgat tcaagtgtaa aagtaaaagg aagaagaaaa 960 aaaaatataa caagtaatgaaaatattatg aatccaaata ataaaagtgt aagtagcatt 1020 aatacaaatc taagtgattcttctaataat aaaaatgata attccttaaa tagtaaaaaa 1080 aatgataata ccttaaatagtaaaaaaaat gataatacct taaatagtaa aaaaaatgat 1140 aataccttaa atagtaaaaataataacaat tccttaaata gtaaaaataa taacaattcc 1200 ttaaatagta aaaagagtaataattcttat aatgacaaac atatagacca tataattcct 1260 gaaggcaaaa ataaaataaacaataatata gatgtaaaac acaatattaa taacaaatta 1320 aatgaaatta atgaagaaccttatgaagat acacataata aagaagagaa ttcttcaaat 1380 aaaaatgata atgatgaaaaaagaaaagaa gaaaataata atataacaag aagatatata 1440 aaaaatgacc aaatgtcatataataatata aatacaaatt ctaatgaata tgacaaaaat 1500 gcatctacat tagatgaaacatatataggt aaaacttttg aaggttatgt ttatagtgtt 1560 aataaaaatg ctgcgtgtattaaattaaaa aatattaata aatatggttt gttatttaaa 1620 aacaaagcaa atttaggtgatgacattgaa gatatgaatg atttttttga aaaagatcaa 1680 ccggttcatg tgaaaatacttggtattaat acaaagaaga atatatttta tctaggaaat 1740 attataaaat ataatgaaaatataaaatta agtaaaggag aatattcaaa gggattaata 1800 acaaaagtat gtgattcctactgttttatt aaagttttaa aaaatggaag cactggatat 1860 ttacataaat caaaattgttttgtatgaat gataaagaaa agaaaaataa 1910 3 2017 DNA Plasmodium falciparum 3acacaatatt aataacaaat taaatgaaat taatgaagaa ccttatgaag atacacataa 60taaagaagag aattcttcaa ataaaaatga taatgatgaa aaaagaaaag aagaaaataa 120taatataaca agaagatata taaaaaatga ccaaatgtca tataataata taaatacaaa 180ttctaatgaa tatgacaaaa atgcatctac attagatgaa acatatatag gtaaaacttt 240tgaaggttat gtttatagtg ttaataaaaa tgctgcgtgt attaaattaa aaaatattaa 300taaatatggt ttgttattta aaaacaaagc aaatttaggt gatgacattg aagatatgaa 360tgattttttt gaaaaagatc aaccggttca tgtgaaaata cttggtatta atacaaagaa 420gaatatattt tatctaggaa atattataaa atataatgaa aatataaaat taagtaaagg 480agaatattca aagggattaa taacaaaagt atgtgattcc tactgtttta ttaaagtttt 540aaaaaatgga agcactggat atttacataa atcaaaattg ttttgtatga atgataaaga 600aaagaaaaat aatgataatc aaaattatga taatccaaat aatgataatc caaattatga 660taatcaaaat tatgataatc aaaattataa taatccaaat tatgatcatc caaattatga 720taatcaaaat tatgataatc aaaattataa taatccaaat aatgattata gtacatcaca 780attatataac agtgataatt tacaaatgga ttttatatat aaattacaat ttacaaaaat 840atttaatata tgggatataa tagatgttga aatattagga actccacaaa atgattataa 900atcaaattat atattgacaa taccaagagg atctaaaaca tttaagaaaa ttttgaatta 960tttgaatgtt ttaaaagaaa atgaagatat taataatata caatataaag gtgattatat 1020ctattctatt gataataata aaaatgacaa tataatagat tcagatataa ataatcatca 1080cattaataat aagaagaaaa agaagaatct atatgatata caaaataata tgaatcattc 1140tccttttaat aagtttcata cagaagatga atatttattt aatgaccatg tccaagaaaa 1200tgttcacacc ttttatgaaa aaaataaaaa atataaaatt acatatgata aagaaaataa 1260tcataaaatg aataaatcgt attatttaaa aaaaaataaa gaattacctt ttaataataa 1320attcaaaaaa atcattaaaa atatttatga ccttcctaac actatttctt tatctatgtt 1380atctaaaaca attaaaatac cattggcatc tataaaaaaa tattttatca tccacgaaaa 1440taaagaatat aattcaagtt ataaaattaa ttcagaacag ataaaacgaa tatgtcaaca 1500tttcaaaata gactgtaatg tagaacagag agatgataat gtggttacaa aagtgaatgg 1560gacaactaag gattgtcaag aaaaagtttt taaaaatgtg acacaggaca gattgaagga 1620aggtgaacag gaacgagtta ttaaggtgga agcaaagatt aaaaatgatg aaatggttat 1680gcaagagcaa aaagatacta aggaggagaa gcacatggac gttcaattta ttgaagaaaa 1740agatattaat gtacaacata ttaatgtaca agatatggat gtacaagata tggatgtaca 1800agatattaat gtacaagata tggatgtgca aaatattaat gtacaagata ttaatattca 1860agatatggat gtgcaaaata ttaataacag tataacactt aacaaatcga caagttgtca 1920aaccgatgaa tcgcgagacg caccgggggg tgaccaaaat gaatcgctcg atgaaaaaga 1980ttcgatggaa aaaagtaaag aaaaaaaaaa aaaaaaa 2017 4 2828 DNA Plasmodiumfalciparum 4 tccaagaaaa tgttcacacc ttttatgaaa aaaataaaaa atataaaattacatatgata 60 aagaaaataa tcataaaatg aataaatcgt attatttaaa aaaaaataaagaattacctt 120 ttaataataa attcaaaaaa atcattaaaa atatttatga ccttcctaacactatttctt 180 tatctatgtt atctaaaaca attaaaatac cattggcatc tataaaaaaatattttatca 240 tccacgaaaa taaagaatat aattcaagtt ataaaattaa ttcagaacagataaaacgaa 300 tatgtcaaca tttcaaaata gactgtaatg tagaacagag agatgataatgtggttacaa 360 aagtgaatgg gacaactaag gattgtcaag aaaaagtttt taaaaatgtgacacaggaca 420 gattgaagga aggtgaacag gaacgagtta ttaaggtgga agcaaagattaaaaatgatg 480 aaatggttat gcaagagcaa aaagatacta aggaggagaa gcacatggacgttcaattta 540 ttgaagaaaa agatattaat gtacaacata ttaatgtaca agatatggatgtacaagata 600 tggatgtaca agatattaat gtacaagata tggatgtgca aaatattaatgtacaagata 660 ttaatattca agatatggat gtgcaaaata ttaataacag tataacacttaacaaatcga 720 caagttgtca aaccgatgaa tcgcgagacg caccgggggg tgaccaaaatgaatcgctcg 780 atgaaaaaga ttcgatggaa aaaagtaaag aaaaaaaaaa aaaaaaagggaaaagtagaa 840 aaaaaaataa agataccaat ttaacattaa aaagtgatag tattcaaaaatcaaagacca 900 ccctagacga taaaaaacga aatgtggtag ttacatttat aggacatattaatcatggaa 960 aaacgtcttt atttgattat atatgtaaaa ccaatgaaca aaaaaaggagtatggactta 1020 taacacaaaa tataagagcg tttaaagcaa ccgtaaggaa taattttacatttactcttg 1080 tcgatacccc aggacatgaa gcatttatgc ctatgagaag tagaggtgttaaaatatcag 1140 atttaagtat tcttgttata tcaggagatg aaggaataca agaacagactgtggaatgta 1200 taaaattaat aaaagaattt aatattaaaa ttattattgc aataactaaagtagatattc 1260 ctaatgttga tgtagataga ataattaatg atttgttata tcatgatataacaacagaat 1320 taaatggagg ggaaatacaa gtagttgaat gttctattta taaagaagaaagtatagata 1380 aattattaga tgccatatat ttagaatctg aatttcttaa tctacaaaccaatcctgata 1440 agaaacatga acaggctcaa ggtgttgttt tagattctta tatagataaaaatggaattg 1500 tttctataaa tttgttacaa aatggtgtat taaatataaa tgatcatttttatactgggt 1560 catcatatgg aaaagtgaag atattaaaag atcatttaaa taaaaatattaaaagtgcat 1620 atccatcgga tcctattaaa attattggat acaacaaaaa ttctgttcctgtagcaggtg 1680 acaaatttta tgttgttgaa aatgaagccc tagccaaaga aattgcggaacataataaga 1740 ataaaatgtt aacaatggaa attaataatt ttacttatga tcagacaaatatgaacaggt 1800 ataaagattt tataatatcc agagaaaata aaattggagg ttcttcaggtatactgggag 1860 aaaataattt aaaaaatgac attgatggta atatgacaag ggatgataatatgacaaggg 1920 atgataatat gacaagtgat gataatatga caagggatgg taatagaacaaataatgaca 1980 atattacaag tgatgataat atgtcaaatg attatgataa aataaaagaaacgaaaatgt 2040 atacaaataa taaatcattt caaaaggatg attttttaaa aatacatttgaataatacaa 2100 acgaaaatgt gattaatatg gatccttcaa cacatatagg aaaaaatgaaataaaaacaa 2160 tatactcaaa ttatattatt aaatgtgata aacaaggaag tatagaagttttgaaaaatt 2220 gtatgcttaa attacaaaaa gaagatagta tatgtaaaat aaaaaacaaaattatatatg 2280 ctgatatagg taatgtaaca tcaagtgata taaaatatgc tacaagttttaatgctacaa 2340 taatagcttt tggtgttaaa ttatcaaatg atattaaagg ttcaaaaaattcaaaaggtt 2400 caaaaaatca taataattat cctattatat attcaaatgt cttatatgaacttatagaaa 2460 atgtggaaaa agaaatggaa aagaaattaa gtaaaaaacc aatgggtgaattaaaaggaa 2520 cagcacaaat tttaaaagtt tttaatatat cgaaacttgg aaaggttgcagggtgtattg 2580 ttaaaaaagg tactatctct ataaatagta atattcgtat tttaagaaatgataaagtta 2640 tttatatggg aaaaatcatt tctattaaaa ttgttaagga agaaaaaacacaagtaacgg 2700 aagctgatga atgtggtata ggttttgata attttttgga ttttgagccaaacgatataa 2760 tcgaagcgta cgaaaattaa atatgaaaaa aaaaaaaaaa aatgtaatgtaaaaaaaaaa 2820 aaaaaaaa 2828 5 496 DNA Plasmodium falciparum 5gttacttcag aaacaataaa taagcatgat tcaagtgtaa aagtaaaagg aagaagaaaa 60aaaaatataa caagtaatga aaatattatg aatccaaata ataaaagtgt aagtagcatt 120aatacaaatc taagtgattc ttctaataat aaaaatgata attccttaaa tagtaaaaaa 180aatgataata ccttaaatag taaaaaaaat gataatacct taaatagtaa aaaaaatgat 240aataccttaa atagtaaaaa taataacaat tccttaaata gtaaaaataa taacaattcc 300ttaaatagta aaaagagtaa taattcttat aatgacaaac atatagacca tataattcct 360gaaggcaaaa ataaaataaa caataatata gatgtaaaac acaatattaa taacaaatta 420aatgaaatta atgaagaacc ttatgaagat acacataata aagaagagaa ttcttcaaat 480aaaaatgata atgatg 496 6 1678 DNA Plasmodium falciparum 6 aaaaaaacaaagaaacaaaa aagagatgtg aataagttta tgatgtgaat aaatttatga 60 tgtgtataaatttatgatgt gaataaattt atgatgtgaa taaatttatg atgtgaataa 120 atttatgatgtgtataaatt tatgatgtga ataaatttat gatgtgaata aatttatgat 180 gtgaataaatttatgatgtg aataaattta tgatgtgtat aaatttatga tgtgtataaa 240 tttatgatgtgtataaattt atgatgtgta tatatttatg atgtgaataa atttatgatg 300 tgtataaatttatgatgtga ataaatttat gatgtgtata aatttatgat gtgtatttat 360 ttttcttttttttagtttta tataattata catacatata tatatatata tatatttata 420 tatatatacaaagggatgtt tctaatatgg cgtttgtata aaaccttatt cttaatattg 480 tatattataattttacaaga atatgtatgt cattcttcta atcatataaa aagtgttaat 540 agatgttttatcccctttat ttcagaatat atatataaac agaaaaacag aaaaaataat 600 atatcgtatgattctagtaa cagtcactac aatgataaaa taataccatg tggtaattat 660 aataaacaattaaaatataa aataaatttt tgtcgtaatt tgtattttaa taataaaaat 720 gaatataatagaatatgctc aaggaacaaa ttaaattttc ataatataca aacagataat 780 acaatatacaaaccaaaaaa gaaatattat gaagttggaa aagtaagaga aaaaataaaa 840 atgtataccctttttaaaga tgacaaaata aacacactga aatgtaatga agaagaatct 900 gttacttcagaaacaataaa taagcatgat tcaagtgtaa aagtaaaagg aagaagaaaa 960 aaaaatataacaagtaatga aaatattatg aatccaaata ataaaagtgt aagtagcatt 1020 aatacaaatctaagtgattc ttctaataat aaaaatgata attccttaaa tagtaaaaaa 1080 aatgataataccttaaatag taaaaaaaat gataatacct taaatagtaa aaaaaatgat 1140 aataccttaaatagtaaaaa taataacaat tccttaaata gtaaaaataa taacaattcc 1200 ttaaatagtaaaaagagtaa taattcttat aatgacaaac atatagacca tataattcct 1260 gaaggcaaaaataaaataaa caataatata gatgtaaaac acaatattaa taacaaatta 1320 aatgaaattaatgaagaacc ttatgaagat acacataata aagaagagaa ttcttcaaat 1380 aaaaatgataatgatgaaaa aagaaaagaa gaaaataata atataacaag aagatatata 1440 aaaaatgaccaaatgtcata taataatata aatacaaatt ctaatgaata tgacaaaaat 1500 gcatctacattagatgaaac atatataggt aaaacttttg aaggttatgt ttatagtgtt 1560 aataaaaatgctgcgtgtat taaattaaaa aatattaata aatatggttt gttatttaaa 1620 aacaaagcaaatttaggtga tgacattgaa gatatgaatg atttttttga aaaagatc 1678 7 1799 DNAPlasmodium falciparum 7 agatattaat aatatacaat ataaaggtga ttatatctattctattgata ataataaaaa 60 tgacaatata atagattcag atataaataa tcatcacattaataataaga agaaaaagaa 120 gaatctatat gatatacaaa ataatatgaa tcattctccttttaataagt ttcatacaga 180 agatgaatat ttatttaatg accatgtcca agaaaatgttcacacctttt atgaaaaaaa 240 taaaaaatat aaaattacat atgataaaga aaataatcataaaatgaata aatcgtatta 300 tttaaaaaaa aataaagaat taccttttaa taataaattcaaaaaaatca ttaaaaatat 360 ttatgacctt cctaacacta tttctttatc tatgttatctaaaacaatta aaataccatt 420 ggcatctata aaaaaatatt ttatcatcca cgaaaataaagaatataatt caagttataa 480 aattaattca gaacagataa aacgaatatg tcaacatttcaaaatagact gtaatgtaga 540 acagagagat gataatgtgg ttacaaaagt gaatgggacaactaaggatt gtcaagaaaa 600 agtttttaaa aatgtgacac aggacagatt gaaggaaggtgaacaggaac gagttattaa 660 ggtggaagca aagattaaaa atgatgaaat ggttatgcaagagcaaaaag atactaagga 720 ggagaagcac atggacgttc aatttattga agaaaaagatattaatgtac aacatattaa 780 tgtacaagat atggatgtac aagatatgga tgtacaagatattaatgtac aagatatgga 840 tgtgcaaaat attaatgtac aagatattaa tattcaagatatggatgtgc aaaatattaa 900 taacagtata acacttaaca aatcgacaag ttgtcaaaccgatgaatcgc gagacgcacc 960 ggggggtgac caaaatgaat cgctcgatga aaaagattcgatggaaaaaa gtaaagaaaa 1020 aaaaaaaaaa aaagggaaaa gtagaaaaaa aaataaagataccaatttaa cattaaaaag 1080 tgatagtatt caaaaatcaa agaccaccct agacgataaaaaacgaaatg tggtagttac 1140 atttatagga catattaatc atggaaaaac gtctttatttgattatatat gtaaaaccaa 1200 tgaacaaaaa aaggagtatg gacttataac acaaaatataagagcgttta aagcaaccgt 1260 aaggaataat tttacattta ctcttgtcga taccccaggacatgaagcat ttatgcctat 1320 gagaagtaga ggtgttaaaa tatcagattt aagtattcttgttatatcag gagatgaagg 1380 aatacaagaa cagactgtgg aatgtataaa attaataaaagaatttaata ttaaaattat 1440 tattgcaata actaaagtag atattcctaa tgttgatgtagatagaataa ttaatgattt 1500 gttatatcat gatataacaa cagaattaaa tggaggggaaatacaagtag ttgaatgttc 1560 tatttataaa gaagaaagta tagataaatt attagatgccatatatttag aatctgaatt 1620 tcttaatcta caaaccaatc ctgataagaa acatgaacaggctcaaggtg ttgttttaga 1680 ttcttatata gataaaaatg gaattgtttc tataaatttgttacaaaatg gtgtattaaa 1740 tataaatgat catttttata ctgggtcatc atatggaaaagtgaagatat taaaagatc 1799 8 693 DNA Plasmodium falciparum 8 ggatccttcaacacatatag gaaaaaatga aataaaaaca atatactcaa attatattat 60 taaatgtgataaacaaggaa gtatagaagt tttgaaaaat tgtatgctta aattacaaaa 120 agaagatagtatatgtaaaa taaaaaacaa aattatatat gctgatatag gtaatgtaac 180 atcaagtgatataaaatatg ctacaagttt taatgctaca ataatagctt ttggtgttaa 240 attatcaaatgatattaaag gttcaaaaaa ttcaaaaggt tcaaaaaatc ataataatta 300 tcctattatatattcaaatg tcttatatga acttatagaa aatgtggaaa aagaaatgga 360 aaagaaattaagtaaaaaac caatgggtga attaaaagga acagcacaaa ttttaaaagt 420 ttttaatatatcgaaacttg gaaaggttgc agggtgtatt gttaaaaaag gtactatctc 480 tataaatagtaatattcgta ttttaagaaa tgataaagtt atttatatgg gaaaaatcat 540 ttctattaaaattgttaagg aagaaaaaac acaagtaacg gaagctgatg aatgtggtat 600 aggttttgataattttttgg attttgagcc aaacgatata atcgaagcgt acgaaaatta 660 aatatgaaaaaaaaaaaaaa aaatgtaatg taa 693 9 1610 PRT Plasmodium falciparum 9 Met PheLeu Ile Trp Arg Leu Tyr Lys Thr Leu Phe Leu Ile Leu Tyr 1 5 10 15 IleIle Ile Leu Gln Glu Tyr Val Cys His Ser Ser Asn His Ile Lys 20 25 30 SerVal Asn Arg Cys Phe Ile Pro Phe Ile Ser Glu Tyr Ile Tyr Lys 35 40 45 GlnLys Asn Arg Lys Asn Asn Ile Ser Tyr Asp Ser Ser Asn Ser His 50 55 60 TyrAsn Asp Lys Ile Ile Pro Cys Gly Asn Tyr Asn Lys Gln Leu Lys 65 70 75 80Tyr Lys Ile Asn Phe Cys Arg Asn Leu Tyr Phe Asn Asn Lys Asn Glu 85 90 95Tyr Asn Arg Ile Cys Ser Arg Asn Lys Leu Asn Phe His Asn Ile Gln 100 105110 Thr Asp Asn Thr Ile Tyr Lys Pro Lys Lys Lys Tyr Tyr Glu Val Gly 115120 125 Lys Val Arg Glu Lys Ile Lys Met Tyr Thr Leu Phe Lys Asp Asp Lys130 135 140 Ile Asn Thr Leu Lys Cys Asn Glu Glu Glu Ser Val Thr Ser GluThr 145 150 155 160 Ile Asn Lys His Asp Ser Ser Val Lys Val Lys Gly ArgArg Lys Lys 165 170 175 Asn Ile Thr Ser Asn Glu Asn Ile Met Asn Pro AsnAsn Lys Ser Val 180 185 190 Ser Ser Ile Asn Thr Asn Leu Ser Asp Ser SerAsn Asn Lys Asn Asp 195 200 205 Asn Ser Leu Asn Ser Lys Lys Asn Asp AsnThr Leu Asn Ser Lys Lys 210 215 220 Asn Asp Asn Thr Leu Asn Ser Lys LysAsn Asp Asn Thr Leu Asn Ser 225 230 235 240 Lys Asn Asn Asn Asn Ser LeuAsn Ser Lys Asn Asn Asn Asn Ser Leu 245 250 255 Asn Ser Lys Lys Ser AsnAsn Ser Tyr Asn Asp Lys His Ile Asp His 260 265 270 Ile Ile Pro Glu GlyLys Asn Lys Ile Asn Asn Asn Ile Asp Val Lys 275 280 285 His Asn Ile AsnAsn Lys Leu Asn Glu Ile Asn Glu Glu Pro Tyr Glu 290 295 300 Asp Thr HisAsn Lys Glu Glu Asn Ser Ser Asn Lys Asn Asp Asn Asp 305 310 315 320 GluLys Arg Lys Glu Glu Asn Asn Asn Ile Thr Arg Arg Tyr Ile Lys 325 330 335Asn Asp Gln Met Ser Tyr Asn Asn Ile Asn Thr Asn Ser Asn Glu Tyr 340 345350 Asp Lys Asn Ala Ser Thr Leu Asp Glu Thr Tyr Ile Gly Lys Thr Phe 355360 365 Glu Gly Tyr Val Tyr Ser Val Asn Lys Asn Ala Ala Cys Ile Lys Leu370 375 380 Lys Asn Ile Asn Lys Tyr Gly Leu Leu Phe Lys Asn Lys Ala AsnLeu 385 390 395 400 Gly Asp Asp Ile Glu Asp Met Asn Asp Phe Phe Glu LysAsp Gln Pro 405 410 415 Val His Val Lys Ile Leu Gly Ile Asn Thr Lys LysAsn Ile Phe Tyr 420 425 430 Leu Gly Asn Ile Ile Lys Tyr Asn Glu Asn IleLys Leu Ser Lys Gly 435 440 445 Glu Tyr Ser Lys Gly Leu Ile Thr Lys ValCys Asp Ser Tyr Cys Phe 450 455 460 Ile Lys Val Leu Lys Asn Gly Ser ThrGly Tyr Leu His Lys Ser Lys 465 470 475 480 Leu Phe Cys Met Asn Asp LysGlu Lys Lys Asn Asn Asp Asn Gln Asn 485 490 495 Tyr Asp Asn Pro Asn AsnAsp Asn Pro Asn Tyr Asp Asn Gln Asn Tyr 500 505 510 Asp Asn Gln Asn TyrAsn Asn Pro Asn Tyr Asp His Pro Asn Tyr Asp 515 520 525 Asn Gln Asn TyrAsp Asn Gln Asn Tyr Asn Asn Pro Asn Asn Asp Tyr 530 535 540 Ser Thr SerGln Leu Tyr Asn Ser Asp Asn Leu Gln Met Asp Phe Ile 545 550 555 560 TyrLys Leu Gln Phe Thr Lys Ile Phe Asn Ile Trp Asp Ile Ile Asp 565 570 575Val Glu Ile Leu Gly Thr Pro Gln Asn Asp Tyr Lys Ser Asn Tyr Ile 580 585590 Leu Thr Ile Pro Arg Gly Ser Lys Thr Phe Lys Lys Ile Leu Asn Tyr 595600 605 Leu Asn Val Leu Lys Glu Asn Glu Asp Ile Asn Asn Ile Gln Tyr Lys610 615 620 Gly Asp Tyr Ile Tyr Ser Ile Asp Asn Asn Lys Asn Asp Asn IleIle 625 630 635 640 Asp Ser Asp Ile Asn Asn His His Ile Asn Asn Lys LysLys Lys Lys 645 650 655 Asn Leu Tyr Asp Ile Gln Asn Asn Met Asn His SerPro Phe Asn Lys 660 665 670 Phe His Thr Glu Asp Glu Tyr Leu Phe Asn AspHis Val Gln Glu Asn 675 680 685 Val His Thr Phe Tyr Glu Lys Asn Lys LysTyr Lys Ile Thr Tyr Asp 690 695 700 Lys Glu Asn Asn His Lys Met Asn LysSer Tyr Tyr Leu Lys Lys Asn 705 710 715 720 Lys Glu Leu Pro Phe Asn AsnLys Phe Lys Lys Ile Ile Lys Asn Ile 725 730 735 Tyr Asp Leu Pro Asn ThrIle Ser Leu Ser Met Leu Ser Lys Thr Ile 740 745 750 Lys Ile Pro Leu AlaSer Ile Lys Lys Tyr Phe Ile Ile His Glu Asn 755 760 765 Lys Glu Tyr AsnSer Ser Tyr Lys Ile Asn Ser Glu Gln Ile Lys Arg 770 775 780 Ile Cys GlnHis Phe Lys Ile Asp Cys Asn Val Glu Gln Arg Asp Asp 785 790 795 800 AsnVal Val Thr Lys Val Asn Gly Thr Thr Lys Asp Cys Gln Glu Lys 805 810 815Val Phe Lys Asn Val Thr Gln Asp Arg Leu Lys Glu Gly Glu Gln Glu 820 825830 Arg Val Ile Lys Val Glu Ala Lys Ile Lys Asn Asp Glu Met Val Met 835840 845 Gln Glu Gln Lys Asp Thr Lys Glu Glu Lys His Met Asp Val Gln Phe850 855 860 Ile Glu Glu Lys Asp Ile Asn Val Gln His Ile Asn Val Gln AspMet 865 870 875 880 Asp Val Gln Asp Met Asp Val Gln Asp Ile Asn Val GlnAsp Met Asp 885 890 895 Val Gln Asn Ile Asn Val Gln Asp Ile Asn Ile GlnAsp Met Asp Val 900 905 910 Gln Asn Ile Asn Asn Ser Ile Thr Leu Asn LysSer Thr Ser Cys Gln 915 920 925 Thr Asp Glu Ser Arg Asp Ala Pro Gly GlyAsp Gln Asn Glu Ser Leu 930 935 940 Asp Glu Lys Asp Ser Met Glu Lys SerLys Glu Lys Lys Lys Lys Lys 945 950 955 960 Gly Lys Ser Arg Lys Lys AsnLys Asp Thr Asn Leu Thr Leu Lys Ser 965 970 975 Asp Ser Ile Gln Lys SerLys Thr Thr Leu Asp Asp Lys Lys Arg Asn 980 985 990 Val Val Val Thr PheIle Gly His Ile Asn His Gly Lys Thr Ser Leu 995 1000 1005 Phe Asp TyrIle Cys Lys Thr Asn Glu Gln Lys Lys Glu Tyr Gly 1010 1015 1020 Leu IleThr Gln Asn Ile Arg Ala Phe Lys Ala Thr Val Arg Asn 1025 1030 1035 AsnPhe Thr Phe Thr Leu Val Asp Thr Pro Gly His Glu Ala Phe 1040 1045 1050Met Pro Met Arg Ser Arg Gly Val Lys Ile Ser Asp Leu Ser Ile 1055 10601065 Leu Val Ile Ser Gly Asp Glu Gly Ile Gln Glu Gln Thr Val Glu 10701075 1080 Cys Ile Lys Leu Ile Lys Glu Phe Asn Ile Lys Ile Ile Ile Ala1085 1090 1095 Ile Thr Lys Val Asp Ile Pro Asn Val Asp Val Asp Arg IleIle 1100 1105 1110 Asn Asp Leu Leu Tyr His Asp Ile Thr Thr Glu Leu AsnGly Gly 1115 1120 1125 Glu Ile Gln Val Val Glu Cys Ser Ile Tyr Lys GluGlu Ser Ile 1130 1135 1140 Asp Lys Leu Leu Asp Ala Ile Tyr Leu Glu SerGlu Phe Leu Asn 1145 1150 1155 Leu Gln Thr Asn Pro Asp Lys Lys His GluGln Ala Gln Gly Val 1160 1165 1170 Val Leu Asp Ser Tyr Ile Asp Lys AsnGly Ile Val Ser Ile Asn 1175 1180 1185 Leu Leu Gln Asn Gly Val Leu AsnIle Asn Asp His Phe Tyr Thr 1190 1195 1200 Gly Ser Ser Tyr Gly Lys ValLys Ile Leu Lys Asp His Leu Asn 1205 1210 1215 Lys Asn Ile Lys Ser AlaTyr Pro Ser Asp Pro Ile Lys Ile Ile 1220 1225 1230 Gly Tyr Asn Lys AsnSer Val Pro Val Ala Gly Asp Lys Phe Tyr 1235 1240 1245 Val Val Glu AsnGlu Ala Leu Ala Lys Glu Ile Ala Glu His Asn 1250 1255 1260 Lys Asn LysMet Leu Thr Met Glu Ile Asn Asn Phe Thr Tyr Asp 1265 1270 1275 Gln ThrAsn Met Asn Arg Tyr Lys Asp Phe Ile Ile Ser Arg Glu 1280 1285 1290 AsnLys Ile Gly Gly Ser Ser Gly Ile Leu Gly Glu Asn Asn Leu 1295 1300 1305Lys Asn Asp Ile Asp Gly Asn Met Thr Arg Asp Asp Asn Met Thr 1310 13151320 Arg Asp Asp Asn Met Thr Ser Asp Asp Asn Met Thr Arg Asp Gly 13251330 1335 Asn Arg Thr Asn Asn Asp Asn Ile Thr Ser Asp Asp Asn Met Ser1340 1345 1350 Asn Asp Tyr Asp Lys Ile Lys Glu Thr Lys Met Tyr Thr AsnAsn 1355 1360 1365 Lys Ser Phe Gln Lys Asp Asp Phe Leu Lys Ile His LeuAsn Asn 1370 1375 1380 Thr Asn Glu Asn Val Ile Asn Met Asp Pro Ser ThrHis Ile Gly 1385 1390 1395 Lys Asn Glu Ile Lys Thr Ile Tyr Ser Asn TyrIle Ile Lys Cys 1400 1405 1410 Asp Lys Gln Gly Ser Ile Glu Val Leu LysAsn Cys Met Leu Lys 1415 1420 1425 Leu Gln Lys Glu Asp Ser Ile Cys LysIle Lys Asn Lys Ile Ile 1430 1435 1440 Tyr Ala Asp Ile Gly Asn Val ThrSer Ser Asp Ile Lys Tyr Ala 1445 1450 1455 Thr Ser Phe Asn Ala Thr IleIle Ala Phe Gly Val Lys Leu Ser 1460 1465 1470 Asn Asp Ile Lys Gly SerLys Asn Ser Lys Gly Ser Lys Asn His 1475 1480 1485 Asn Asn Tyr Pro IleIle Tyr Ser Asn Val Leu Tyr Glu Leu Ile 1490 1495 1500 Glu Asn Val GluLys Glu Met Glu Lys Lys Leu Ser Lys Lys Pro 1505 1510 1515 Met Gly GluLeu Lys Gly Thr Ala Gln Ile Leu Lys Val Phe Asn 1520 1525 1530 Ile SerLys Leu Gly Lys Val Ala Gly Cys Ile Val Lys Lys Gly 1535 1540 1545 ThrIle Ser Ile Asn Ser Asn Ile Arg Ile Leu Arg Asn Asp Lys 1550 1555 1560Val Ile Tyr Met Gly Lys Ile Ile Ser Ile Lys Ile Val Lys Glu 1565 15701575 Glu Lys Thr Gln Val Thr Glu Ala Asp Glu Cys Gly Ile Gly Phe 15801585 1590 Asp Asn Phe Leu Asp Phe Glu Pro Asn Asp Ile Ile Glu Ala Tyr1595 1600 1605 Glu Asn 1610 10 951 DNA Plasmodium falciparum 10atgtttctaa tatggcgttt gtataaaacc ttattcttaa tattgtatat tataatttta 60caagaatatg tatgtcattc ttctaatcat ataaaaagtg ttaatagatg ttttatcccc 120tttatttcag aatatatata taaacagaaa aacagaaaaa ataatatatc gtatgattct 180agtaacagtc actacaatga taaaataata ccatgtggta attataataa acaattaaaa 240tataaaataa atttttgtcg taatttgtat tttaataata aaaatgaata taatagaata 300tgctcaagga acaaattaaa ttttcataat atacaaacag ataatacaat atacaaacca 360aaaaagaaat attatgaagt tggaaaagta agagaaaaaa taaaaatgta tacccttttt 420aaagatgaca aaataaacac actgaaatgt aatgaagaag aatctgttac ttcagaaaca 480ataaataagc atgattcaag tgtaaaagta aaaggaagaa gaaaaaaaaa tataacaagt 540aatgaaaata ttatgaatcc aaataataaa agtgtaagta gcattaatac aaatctaagt 600gattcttcta ataataaaaa tgataattcc ttaaatagta aaaaaaatga taatacctta 660aatagtaaaa aaaatgataa taccttaaat agtaaaaaaa atgataatac cttaaatagt 720aaaaataata acaattcctt aaatagtaaa aataataaca attccttaaa tagtaaaaag 780agtaataatt cttataatga caaacatata gaccatataa ttcctgaagg caaaaataaa 840ataaacaata atatagatgt aaaacacaat attaataaca aattaaatga aattaatgaa 900gaaccttatg aagatacaca taataaagaa gagaattctt caaataaaaa a 951 11 978 DNAPlasmodium falciparum 11 atgtttctaa tatggcgttt gtataaaacc ttattcttaatattgtatat tataatttta 60 caagaatatg tatgtcattc ttctaatcat ataaaaagtgttaatagatg ttttatcccc 120 tttatttcag aatatatata taaacagaaa aacagaaaaaataatatatc gtatgattct 180 agtaacagtc actacaatga taaaataata ccatgtggtaattataataa acaattaaaa 240 tataaaataa atttttgtcg taatttgtat tttaataataaaaatgaata taatagaata 300 tgctcaagga acaaattaaa ttttcataat atacaaacagataatacaat atacaaacca 360 aaaaagaaat attatgaagt tggaaaagta agagaaaaaataaaaatgta tacccttttt 420 aaagatgaca aaataaacac actgaaatgt aatgaagaagaatctgttac ttcagaaaca 480 ataaataagc atgattcaag tgtaaaagta aaaggaagaagaaaaaaaaa tataacaagt 540 aatgaaaata ttatgaatcc aaataataaa agtgtaagtagcattaatac aaatctaagt 600 gattcttcta ataataaaaa tgataattcc ttaaatagtaaaaaaaatga taatacctta 660 aatagtaaaa aaaatgataa taccttaaat agtaaaaaaaatgataatac cttaaatagt 720 aaaaaaaatg ataatacctt aaatagtaaa aataataacaattccttaaa tagtaaaaat 780 aataacaatt ccttaaatag taaaaagagt aataattcttataatgacaa acatatagac 840 catataattc ctgaaggcaa aaataaaata aacaataatatagatgtaaa acacaatatt 900 aataacaaat taaatgaaat taatgaagaa ccttatgaagatacacataa taaagaagag 960 aattcttcaa ataaaaaa 978 12 978 DNA Plasmodiumfalciparum 12 atgtttctaa tatggcgttt gtataaaacc ttattcttaa tattgtatattataatttta 60 caagaatatg tatgtcattc ttctaatcat ataaaaagtg ttaatagatgttttatcccc 120 tttatttcag aatatatata taaacagaaa aacagaaaaa ataatatatcgtatgattct 180 agtaacagtc actacaatga taaaataata ccatgtggta attataataaacaattaaaa 240 tataaaataa atttttgtcg taatttgtat tttaataata aaaatgaatataatagaata 300 tgctcaagga acaaattaaa ttttcataat atacaaacag ataatacaatatacaaacca 360 aaaaagaaat attatgaagt tggaaaagta agagaaaaaa taaaaatgtatacccttttt 420 aaagatgaca aaataaacac actgaaatgt aatgaagaag aatctgttacttcagaaaca 480 ataaataagc atgattcaag tgtaaaagta aaaggaagaa gaaaaaaaaatataacaagt 540 aatgaaaata ttatgaatcc aaataataaa agtgtaagta gcattaatacaaatctaagt 600 gattcttcta ataataaaaa tgataattcc ttaaatagta aaaaaaatgataatacctta 660 aatagtaaaa aaaatgataa taccttaaat agtaaaaaaa atgataataccttaaatagt 720 aaaaaaaatg ataatacctt aaatagtaaa aataataaca attccttaaatagtaaaaat 780 aataacaatt ccttaaatag taaaaagagt aataattctt ataatgacaaacatatagac 840 catataattc ctgaaggcaa aaataaaata aacaataata tagatgtaaaacacaatatt 900 aataacaaat taaatgaaat taatgaagaa ccttatgaag atacacataataaagaagag 960 aattcttcaa ataaaaaa 978

What is claimed is:
 1. A purified polypeptide that has an amino acidsequence that is set forth in SEQ ID NO
 9. 2. The purified polypeptideof claim 1, wherein the polypeptide is a recombinant polypeptide.
 3. Apurified polypeptide that is capable of eliciting an immune responseagainst Plasmodium, wherein the polypeptide has an amino acid sequencethat is set forth in SEQ ID NO
 9. 4. The purified polypeptide of claim3, wherein the polypeptide comprises at least 50 contiguous amino acidresidues.
 5. A pharmaceutical composition for eliciting an immuneresponse against Plasmodium, comprising a polypeptide having an aminoacid sequence that is set forth in SEQ ID NO 9 and a pharmaceuticallyacceptable carrier.
 6. The pharmaceutical composition of claim 5,wherein the polypeptide comprises at least 50 contiguous amino acidresidues.
 7. The pharmaceutical composition of claim 5, wherein thepolypeptide is encoded by a polynucleotide according to SEQ ID NO
 1. 8.A method of eliciting an immune response in a subject againstPlasmodium, comprising administering to the subject an isolatedpolypeptide, wherein the polypeptide has an amino acid sequence that isset forth in SEQ ID NO
 9. 9. The method according to claim 8, whereinthe polypeptide is encoded by a polynucleotide according to SEQ ID NO 1.10. The method according to claim 8, wherein the isolated polypeptidecomprises at least 50 contiguous amino acid residues.
 11. The methodaccording to claim 8, wherein the isolated polypeptide is a recombinantpolypeptide.
 12. A natural, synthetic or recombinant DNA or RNA vaccinehaving a nucleotide sequence SEQ ID NO. 1, wherein the vaccine iscapable of eliciting development of anti-Plasmodium antibodies.
 13. Anatural, synthetic or recombinant DNA or RNA vaccine having a nucleotidesequence SEQ ID NO. 2, wherein the vaccine is capable of elicitingdevelopment of anti-Plasmodium antibodies.
 14. A natural, synthetic orrecombinant DNA or RNA vaccine having a nucleotide sequence SEQ ID NO.3, wherein the vaccine is capable of eliciting development ofanti-Plasmodium antibodies.
 15. A natural, synthetic or recombinant DNAor RNA vaccine having a nucleotide sequence SEQ ID NO. 4, wherein thevaccine is capable of eliciting development of anti-Plasmodiumantibodies.
 16. A natural, synthetic or recombinant DNA or RNA vaccinehaving a nucleotide sequence SEQ ID NO. 5, wherein the vaccine iscapable of eliciting development of anti-Plasmodium antibodies.
 17. Anatural, synthetic or recombinant DNA or RNA vaccine having a nucleotidesequence SEQ ID NO. 6, wherein the vaccine is capable of elicitingdevelopment of anti-Plasmodium antibodies.
 18. A natural, synthetic orrecombinant DNA or RNA vaccine having a nucleotide sequence SEQ ID NO.7, wherein the vaccine is capable of eliciting development ofanti-Plasmodium antibodies.
 19. A natural, synthetic or recombinant DNAor RNA vaccine having a nucleotide sequence SEQ ID NO. 8, wherein thevaccine is capable of eliciting development of anti-Plasmodiumantibodies.
 20. A method for the in vitro diagnosis of malaria, whichcomprises contacting a body specimen taken from an individual with thepurified polypeptide of claim 3 under conditions suitable for bindingbetween the polypeptide and antibodies present in the body specimen;detecting binding between the polypeptide and antibodies; andcorrelating the binding with the presence of malaria.
 21. A kit for invitro diagnosis of malaria comprising: the purified polypeptideaccording to claim 3; a medium suitable for formation of anantigen-antibody complex; and reagents for detection of theantigen-antibody complex.
 22. A kit for in vitro diagnosis of malariacomprising a set of PCR primers suitable for amplification of MB2.
 23. Akit for in vitro diagnosis of malaria comprising: a purified DNA thathas a nucleotide sequence that is set forth in SEQ ID NO 1; and reagentsfor detecting complementary DNA or RNA from a body specimen.
 24. Anantibody that binds specifically to MB2 polypeptide.
 25. A method ofmaking an antibody, comprising immunizing a non-human animal with animmunogenic fragment of MB2 polypeptide.
 26. A method of making anantibody, comprising providing a hybridoma cell that produces amonoclonal antibody specific for MB2 polypeptide, and culturing the cellunder conditions that permit production of the monoclonal antibody. 27.A method of inhibiting Plasmodium infection in a patient, comprisingadministering to the patient a composition comprising the antibody ofclaim
 24. 28. A method of determining whether a biological samplecontains Plasmodium, comprising contacting the sample with the antibodyof claim 24 and determining whether the antibody specifically binds tothe sample, said binding being an indication that the sample containsPlasmodium.
 29. The method of claim 28, wherein the biological sample isa mosquito.
 30. The method of claim 28, wherein the biological sample isa human body specimen.
 31. A method for purifying MB2 polypeptide from abiological sample containing MB2 polypeptide, comprising providing anaffinity matrix comprising the antibody of claim 24 bound to a solidsupport; contacting the biological sample with the affinity matrix, toproduce an affinity matrix-MB2 polypeptide complex; separating theaffinity matrix-MB2 polypeptide complex from the remainder of thebiological sample; and releasing MB2 polypeptide from the affinitymatrix.
 32. A composition comprising purified antigen MB2.
 33. A methodof eliciting an immune response in an animal, comprising introducinginto the animal a composition comprising purified antigen MB2.
 34. Amethod of generating antibodies specific for antigen MB2, comprisingintroducing into an animal a composition comprising purified antigenMB2.
 35. A live bacterial cell vector that (a) infects a human and (b)is stably transformed with, and expresses, a heterologous DNA encodingantigen MB2.
 36. A kit for in vitro detection of Plasmodium comprising:the antibody of claim 24; a medium suitable for formation of anantigen-antibody complex; and reagents for detection of theantigen-antibody complex.
 37. A kit for in vitro detection of Plasmodiumcomprising a set of PCR primers suitable for amplification of MB2.
 38. Akit for in vitro detection of Plasmodium in a sample comprising:purified DNA that has a nucleotide sequence that is set forth in SEQ IDNO 1; and reagents for detecting complementary DNA or RNA from thesample.